O-Linked Glycoforms Of Polypeptides And Method To Manufacture Them

ABSTRACT

The present invention relates to compositions comprising glycoproteins having altered patterns of O-linked glycosylation, in particular Factor VII, Factor IX, and methods for making these.

FIELD OF THE INVENTION

The present invention relates to compositions comprising glycoproteinshaving altered patterns of O-linked glycosylation, in particular FactorVII, Factor IX and other blood clotting factors.

BACKGROUND OF THE INVENTION

The biological activity of many glycoproteins is highly dependent uponthe presence or absence of particular oligosaccharide structuresattached to the glycoprotein. The glycosylation pattern of a therapeuticglycoprotein can affect numerous aspects of the therapeutic efficacy,such as, e.g, solubility, resistance to proteolytic attack, thermalinactivation, immunogenicity, half-life, bioactivity, bioavailability,and stability.

Glycosylation is a complex post-transitional modification that is celldependent. Following translation, proteins are transported into theendoplasmic reticulum (ER), glycosylated and sent to the Golgi forfurther processing and subsequent targeting and/or secretion. Duringglycosylation, either N-linked or O-linked glycoproteins are formed.

Serum proteins involved in coagulation or fibrinolysis, including, e.g.,Factor VII and Factor IX are proving to be useful therapeutic agents totreat a variety of pathological conditions. Accordingly, there is anincreasing need for formulations comprising these proteins that arepharmaceutically acceptable and exhibit a uniform and predeterminedclinical efficacy.

Because of the many disadvantages of using human plasma as a source ofpharmaceutical products, it is preferred to produce these proteins inrecombinant systems. The clotting proteins, however, are subject to avariety of co- and posttranslational modifications, including, e.g.,asparagine-linked (N-linked) glycosylation; serine- or threonine-linked(O-linked) glycosylation; and T-carboxylation of glu residues. Thesemodifications may be qualitatively or quantitatively different whenheterologous cells are used as hosts for large-scale production of theproteins. In particular, production in heterologous cells often resultsin a different array of glycoforms, which identical polypeptides arehaving different covalently linked oligosaccharide structures.

In different systems, variations in the oligosaccharide structure oftherapeutic proteins have been linked to, inter alia, changes inimmunogenicity and in vivo clearance. Thus, there is a need in the artfor compositions and methods that provide glycoprotein preparations,particularly preparations comprising recombinant Factor IX orrecombinant human Factor VII or modified Factor VII or FactorVII-related polypeptides that contain predetermined glycoform patterns.

SUMMARY OF THE INVENTION

The present invention relates to preparations comprising polypeptidesthat exhibit predetermined serine or threonine-linked glycoformpatterns. The preparations are at least about 80% homogenous in respectof the attached glycans or oligosaccharide chains, preferably at leastabout 90%, at least about 95%, or at least about 98% homologous.

As used herein, a glycoform pattern refers to the distribution withinthe preparation of oligosaccharide chains having varying structures thatare covalently linked to a serine or threonine residue located in anEGF-like domain in the amino acid backbone of the polypeptide.

In one aspect, the invention provides a preparation of a glycoproteincontaining a Cys-X1-Ser/Thr-X2-Pro-Cys motif and wherein saidserine/threonine forms part of a Glc-O-Ser/Thr covalent bond, saidpreparation containing a substantially uniform serine/threonine-linkedglycosylation pattern.

In one embodiment of the invention, the glycosylation pattern is atleast 80% uniform, preferably at least 85%, at least 90%, at least 95%,or at least 98% uniform.

In one embodiment, the serine/threonine-linked glycans are Xyl-Xyl-Glc-;in another, the glycans are Xyl-Glc-; in yet another, the glycans areGlc-.

In different embodiments the glycoproteins are selected from the groupof: Factor VII polypeptides, Factor VII-related polypeptides, Factor IXpolypeptides, Factor X polypeptides, Factor XII polypeptides, andprotein Z polypeptides. In a preferred embodiment, the glycoprotein isselected from the group of: Human Factor VII, Factor VII sequencevariants, human Factor IX, and Factor IX sequence variants. In oneembodiment, the glycoprotein is a Factor VII variant wherein the ratiobetween the activity of the Factor VII-variant and the activity ofnative human factor VIIa (wild-type FVIIa) is at least about 1.25 whentested in the “In Vitro Hydrolysis Assay” as described in the pre-sentdescription, preferably at least about 2.0, or at least about 4.0.

In another aspect the invention provides methods for making preparationsof glycoproteins containing Cys-X1-Ser/Thr-X2-Pro-Cys motifs and whereinsaid serine/threonine forms part of a Glc-O-Ser/Thr covalent bond, saidpreparations containing a substantially uniform serine/threonine-linkedglycosylation pattern. The methods are useful for remodelling oraltering the glycosylation pattern present on a glycoprotein upon itsinitial expression.

More particular, the present invention provide a general enzymaticmethodology for the modification of glycans (in particular O-linkedglycans) of glycoproteins, in order to improve or enhance theirpharmaceutically properties. One method involves treatment of theglycoprotein with xylosidases in order to remove any terminal xyloseresidues; other methods includes attachment of xylose residues to theexposed glucose or xylose residues on the glycoprotein by treatment withxylosyltransferases; a third method includes attachment of glucoseresidues to serine and/or threonine amino acid residues in thepolypeptide backbone thereby creating a glycosylated polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the serine 52 glycosylation of wt-Factor VII.

FIG. 2 shows an O-glycosylation mapping of Factor VII.

FIG. 3 shows a reaction scheme for the making of a preparation ofglycoproteins exhibiting a predetermined serine/threonine-linkedglycosylation.

FIG. 4 shows a chromatogram from first HIC cycle showing fractions “A”and “B”.

FIG. 5 shows a chromatogram obtained by reloading fraction “A” onto theHIC column; Glc-O-Ser52-FVII was identified in the peak fraction,fraction 10.

FIG. 6 shows a chromatogram obtained by reloading fraction “B” onto theHIC column; Xyl-Xyl-Glc-O-Ser52-FVII was identified in the peakfraction, fraction 15.

FIG. 7A shows a tryptic peptide map of the peak fraction, fraction 10;the arrow indicates the Glc-O-Ser52 O-glycopeptide.

FIG. 7B shows a tryptic peptide map of the peak fraction, fraction 15;the arrow indicates the Xyl-Xyl-Glc-O-Ser52 O-glycopeptide.

FIG. 8A shows a total mass analysis of the peak fraction, fraction 10;the arrow indicates the Glc-O-Ser52-rFVIIa O-glycoform.

FIG. 8B shows a total mass analysis of the peak fraction, fraction 15;the arrow indicates the Xyl-Xyl-Glc-O-rFVIIa O-glycoform.

DETAILED DESCRIPTION

The following abbreviations are used herein:

Glc=glucosyl

Xyl=xylosyl

Ser=serine (one letter code: S)

Thr=threonine (one letter code: T)

Pro=proline (one letter code: P)

Cys=cysteine (one letter code: C)

FVII=Factor VII

FVIIa=activated (two-chain) Factor VII

FIX=Factor IX

FIXa=activated (two-chain) Factor IX

As used herein, a “glycoform pattern” (or “glycosylation pattern”)refers to the distribution within the preparation of oligosaccharidechains having varying structures that are covalently linked to a serineor threonine residue located in the amino acid backbone of thepolypeptide.

“Homogeneity” refers to the structural consistency across a populationof polypeptides with conjugated glycans. Thus, a glycoproteinpreparation is said to be about 100% homologous if all containedglycoprotein molecules contain identical glycans attached to therelevant glycosylation site. For example, a preparation of Factor VIIpolypeptides is said to be at least 90% homologous if at least 90% ofthe Factor VII polypeptide molecules contain the glycan of interestattached to serine 52 (e.g., Xyl-Xyl-Glc-O-Ser52).

“Substantially uniform glycoform” or “substantially uniformglycosylation” or “substantially uniform glycosylation pattern”, whenreferring to a glycopeptide species, refers to the percentage ofacceptor moieties, i.e., serine or threonine residues, that areglycosylated by the glycan of interest. For example, in the case ofFactor VII, a substantially uniform glycosylation patterns exists ifsubstantially all (as defined below) of the serine residues in position52 are glycosylated with the glycan of interest. It is understood by oneskilled in the art that the starting material may contain glycosylatedserine and/or threonine residues that are glycosylated with a specieshaving the same structure as the glycan of interest. Thus, thecalculated percent glycosylation includes serine/threonine residues thatare glycosylated with the glycan of interest according to the invention,as well as those serine/threonine residues already glycosylated with theglycan of interest in the starting material.

The term “substantially” is intended to mean that at least about 80%,such as at least about 90%, at least about 95%, or at least about 98% ofthe serine/threonine residues in the glycoprotein is glycosylated with apredetermined, specific glycan or glycan of interest. The glycosylationpattern is typically determined by one or more methods known to thoseskilled in the art, such as, e.g., tryptic digestion followed by highperformance liquid chromatography (HPLC), liquid-chromatography-massspectrometry (LCMS), matrix assisted laser desorption mass time offlight spectrometry (MALDITOF), capillary electrophoresis, and the like.

The term “acceptor moiety” is intended to encompass the group or moietyto which a desired oligo- or mono-saccharide group is transferred suchas, without limitation, the serine/threonine residue located within aCys-X1-Ser/Thr-X2-Pro-Cys motif, a Glc-residue covalently linked to sucha serine/threonine residue, or a Xyl-residue covalently linked to aGlc-residue or a Xyl-residue in a Glc-O-Ser/Thr or Xyl-Glc-O-Ser/Thrmoiety, respectively.

The term “saccharide donor moiety” is intended to encompass an activatedsaccharide donor molecule (e.g., a desired oligo- or mono-saccharidestructure such as, for example, a xylosyl-xylosyl-donor, xylosyl-donor,or glycosyl-donor) having a leaving group (e.g., xylose-UDP orglucose-UDP) suitable for the donor moiety acting as a substrate for therelevant catalysing enzyme (e.g. glycosyltransferase, xylosidase orxylosyltransferase).

Oligosaccharides are considered to have a reducing and a non-reducingend, whether or not the saccharide at the reducing end is in fact areducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right (e.g., Xyl-Xyl-Glc-O-Ser)

EGF Domain-Containing Polypeptides

The term “EGF domain-containing polypeptides” is intended to encompasspeptides, oligopeptides and polypeptides containing one or moreepidermal growth factor (EGF)-like domain(s). EGF domains or repeats aresmall motifs with about 40 amino acids defined by 6 conserved cysteinesforming three disulfide bonds. EGF domain-containing polypeptides allcontain a consensus sequence for O-glucose modification:Cys1-X1-Ser/Thr-X2-Pro-Cys2 (i.e., a Cys1-X1-Ser-X2-Pro-Cys2 or aCys1-X1-Thr-X2-Pro-Cys2 consensus sequence) where Cys1 and Cys2 are thefirst and second conserved cysteines of the EGF repeat and X1 and X2independently is any amino acid.

The term “glycoprotein” is intended to encompass EGF domain-containingpolypeptides containing one or more glycans attached to one or moreserine/threonine amino acid residues of the EGF-domain located in theback bone amino acid sequence of the polypeptide.

As used herein, the term “glycan” or, interchangeable, “sugar chain”,“oligosaccharide chain” or “oligosaccharide moiety” refers to the entireoligosaccharide structure that is covalently linked to a singleserine/threonine residue. The glycan may comprise one or more saccharideunits; examples of glycans include, e.g., Glc-, Xyl-Glc-, andXyl-Xyl-Glc-.

The term “O-glycosylation site” is intended to indicate theglycosylation site at serine/threonine (i.e., Ser or Thr) located withinthe motif Cys1-X1-Ser/Thr-X2-Pro-Cys2 where Cys1 and Cys2 are the firstand second conserved cysteines of the EGF repeat and X1 and X2independently is any amino acid. These include the glycosylation site atposition Ser-52 (552) of human wt-FVII and the corresponding residues inhomologous polypeptides such as, without limitation, FVII sequencevariants and FIX polypeptides. The term “corresponding residues” isintended to indicate the Ser or Thr amino acid residue corresponding tothe Ser52 residue of wild-type Factor VII (see FIG. 1) when thesequences are aligned. Amino acid sequence homology/identity isconveniently determined from aligned sequences, using a suitablecomputer program for sequence alignment, such as, e.g., the ClustalWprogram, version 1.8, 1999 (Thompson et al., 1994, Nucleic AcidResearch, 22: 4673-4680). For example, the wt-factor VII Ser52-residuecorresponds to the Ser53-residue of wt-Factor IX. It is further to beunderstood that polypeptide variants may be created containingnon-naturally occurring Cys-X1-Ser/Thr-X2-Pro-Cys motifs and therebycontaining non-naturally occurring O-glycosylation sites that can beglycosylated in accordance with the present invention. In one embodimentof the invention, the O-glycosylation site is a serine-glycosylationsite and the motif is Cys1-X1-Ser-X2-Pro-Cys2. In another embodiment,the O-glycosylation site is a threonine-glycosylation site and the motifis Cys1-X1-Thr-X2-Pro-Cys2.

The term “terminal glucose” is intended to encompass glucose residueslinked as the terminal sugar residue in a glycan, or oligosaccharidechain, i.e., the terminal sugar of each antenna is glucose. The term“terminal xylose” is intended to encompass xylose residues linked as theterminal sugar residue in a glycan, or oligosaccharide chain.

Enzymes

Protein O-glycosyltransferase may be prepared as described, e.g., inShao et al. (Glycobiology 12(11): 763-770 (2002)).

The alpha-xylosidase enzymes may be prepared, e.g., as described byMonroe et al. (Plant Physiology and Biochemistry 41:877-885 (2003)).

The enzyme, UDP-D-xylose: β-D-glucoside α-1,3-D-xylosyltransferase canbe prepared from HepG2 cells as described by Omichi et al. (Eur. J.Biochem. 245:143-146 (1997)).

The enzyme, UDP-D-xylose: α-D-xyloside α1,3-xylosyltransferase can beprepared from HepG2 cells as described by Minamida et al. ((J. Biochem.(Tokyo) 120: 1002-1006 (1996)).

UDP-beta-D-glucose is commercially available from, e.g., Sigma (SigmaU4625)

UDP-D-xylose is commercially available from, e.g., Sigma (Sigma U5875)

Glycoproteins

The motif: Cys-X1-Ser/Thr-X2-Pro-Cys appears to be primarily found inepidermal growth factor (EGF) domains of multi-modular proteins such ascoagulation and fibrinolytic factors. The motif is a consensus sequencefor O-glucose modification whereby a serine-glucose (Glc-O-Ser) orthreonine-glucose (Glc-O-Thr) bond is formed. Coagulation factors VII,IX, X and XII as well as plasma Protein Z, Fibrillin and thrombospondinhave all been shown to contain the Cys-X1-Ser/Thr-X2-Pro-Cys consensussequence. Of these, Factors VII and IX and Protein Z have been describedto contain the consensus sequence Cys-X1-Ser-X2-Pro-Cys.

The thrombospondins are a family of extracellular proteins thatparticipate in cell-to-cell and cell-to-matrix communication. Theproteins are secreted from platelets. They regulate cellular phenotypeduring tissue genesis and repair.

Protein Z is a vitamin k-dependent plasma protein whose structure issimilar to that of Factors VII, IX and X. In contrast to these proteins,however, Protein Z is not the zymogen of a serine protease because itlacks the His and Ser residues of the catalytic triad. Like Proteins Cand S, Protein Z participates in limiting the coagulation response,believably by assisting in inhibition of activated Factor X (FXa).

Factor X (Stuart Prower Factor) is a vitamin K-dependent serine proteasewhich participates in the blood clotting process by participating inactivation of prothrombin into thrombin.

Factor XII (Hageman factor) is a blood coagulation factor activated bycontact with the sub-endothelial surface of an injured vessel. Alongwith prekallikrein, it serves as the contact factor that initiates theintrinsic pathway of blood coagulation. Kallikrein activates factor XIIto XIIa.

Factor IX (Christmas factor) is a vitamin K-dependent serine proteasewhich participates in the blood clotting process by participating inactivation of FX into FXa.

Factor VII (proconvertin) is a vitamin K-dependent serine protease whichparticipates in the blood clotting process by participating inactivation of prothrombin into thrombin. FVII is activated into FVIIa bycontact with exposed tissue factor (TF) at sites of injury of the vesselwall.

Factor VII Polypeptides and Factor VII-Related Polypeptides

As used herein, the terms “Factor VII polypeptide” or “FVII polypeptide”means any protein comprising the amino acid sequence 1-406 of wild-typehuman Factor VIIa (i.e., a polypeptide having the amino acid sequencedisclosed in U.S. Pat. No. 4,784,950), variants thereof as well asFactor VII-related polypeptides, Factor VII derivatives and Factor VIIconjugates. This includes FVII variants, Factor VII-relatedpolypeptides, Factor VII derivatives and Factor VII conjugatesexhibiting substantially the same or improved biological activityrelative to wild-type human Factor VIIa.

The term “Factor VII” is intended to encompass Factor VII polypeptidesin their uncleaved (zymogen) form, as well as those that have beenproteolytically processed to yield their respective bioactive forms,which may be designated Factor VIIa. Typically, Factor VII is cleavedbetween residues 152 and 153 to yield Factor VIIa. Such variants ofFactor VII may exhibit different properties relative to human FactorVII, including stability, phospholipid binding, altered specificactivity, and the like.

As used herein, “wild type human FVIIa” is a polypeptide having theamino acid sequence disclosed in U.S. Pat. No. 4,784,950.

As used herein, “Factor VII-related polypeptides” encompassespolypeptides, including variants, in which the Factor VIIa biologicalactivity has been substantially modified, such as reduced, relative tothe activity of wild-type Factor VIIa. These polypeptides include,without limitation, Factor VII or Factor VIIa into which specific aminoacid sequence alterations have been introduced that modify or disruptthe bioactivity of the polypeptide.

The term “Factor VII derivative” as used herein, is intended todesignate a FVII polypeptide exhibiting substantially the same orimproved biological activity relative to wild-type Factor VII, in whichone or more of the amino acids of the parent peptide have beengenetically and/or chemically and/or enzymatically modified, e.g. byalkylation, glycosylation, PEGylation, acylation, ester formation oramide formation or the like. This includes but is not limited toPEGylated human Factor VIIa, cysteine-PEGylated human Factor VIIa andvariants thereof. Non-limiting examples of Factor VII derivativesincludes GlycoPegylated FVII derivatives as disclosed in WO 03/31464 andUS Patent applications US 20040043446, US 20040063911, US 20040142856,US 20040137557, and US 20040132640 (Neose Technologies, Inc.); FVIIconjugates as disclosed in WO 01/04287, US patent application20030165996, WO 01/58935, WO 03/93465 (Maxygen ApS) and WO 02/02764, USpatent application 20030211094 (University of Minnesota).

The term “improved biological activity” refers to FVII polypeptides withi) substantially the same or increased proteolytic activity compared torecombinant wild type human Factor VIIa or ii) to FVII polypeptides withsubstantially the same or increased TF binding activity compared torecombinant wild type human Factor VIIa or iii) to FVII polypeptideswith substantially the same or increased half life in blood plasmacompared to recombinant wild type human Factor VIIa. The term “PEGylatedhuman Factor VIIa” means human Factor VIIa, having a PEG moleculeconjugated to a human Factor VIIa polypeptide. It is to be understood,that the PEG molecule may be attached to any part of the Factor VIIapolypeptide including any amino acid residue or carbohydrate moiety ofthe Factor VIIa polypeptide. The term “cysteine-PEGylated human FactorVIIa” means Factor VIIa having a PEG molecule conjugated to a sulfhydrylgroup of a cysteine introduced in human Factor VIIa.

Non-limiting examples of Factor VII variants having substantially thesame or increased proteolytic activity compared to recombinant wild typehuman Factor VIIa include S52A-FVIIa, S60A-FVIIa (Lino et al., Arch.Biochem. Biophys. 352: 182-192, 1998); FVIIa variants exhibitingincreased proteolytic stability as disclosed in U.S. Pat. No. 5,580,560;Factor VIIa that has been proteolytically cleaved between residues 290and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol.Bioeng. 48:501-505, 1995); oxidized forms of Factor VIIa (Kornfelt etal., Arch. Biochem. Biophys. 363:43-54, 1999); FVII variants asdisclosed in PCT/DK02/00189 (corresponding to WO 02/077218); and FVIIvariants exhibiting increased proteolytic stability as disclosed in WO02/38162 (Scripps Research Institute); FVII variants having a modifiedGla-domain and exhibiting an enhanced membrane binding as disclosed inWO 99/20767, U.S. Pat. No. 6,017,882 and U.S. Pat. No. 6,747,003, USpatent application 20030100506 (University of Minnesota) and WO00/66753, US patent applications US 20010018414, US 2004220106, and US200131005, U.S. Pat. No. 6,762,286 and U.S. Pat. No. 6,693,075(University of Minnesota); and FVII variants as disclosed in WO01/58935, U.S. Pat. No. 6,806,063, US patent application 20030096338(Maxygen ApS), WO 03/93465 (Maxygen ApS), WO 04/029091 (Maxygen ApS), WO04/083361 (Maxygen ApS), and WO 04/111242 (Maxygen ApS), as well as inWO 04/108763 (Canadian Blood Services).

Non-limiting examples of FVII variants having increased biologicalactivity compared to wild-type FVIIa include FVII variants as disclosedin WO 01/83725, WO 02/22776, WO 02/077218, PCT/DK02/00635 (correspondingto WO 03/027147), Danish patent application PA 2002 01423 (correspondingto WO 04/029090), Danish patent application PA 2001 01627 (correspondingto WO 03/027147); WO 02/38162 (Scripps Research Institute); and FVIIavariants with enhanced activity as disclosed in JP 2001061479(Chemo-Sero-Therapeutic Res Inst.). Examples of variants of factor VIIinclude, without limitation, L305V-FVII, L305V/M306D/D309S-FVII,L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII,V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII,L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII,V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII,K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII,V158D/M298K-FVII, and S336G-FVII, L305V/K337A-FVII, L305V/V158D-FVII,L305V/E296V-FVII, L305V/M298Q-FVII, L305V/V158T-FVII,L305V/K337A/V158T-FVII, L305V/K337A/M298Q-FVII, L305V/K337A/E296V-FVII,L305V/K337A/V158D-FVII, L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVII,L305V/V158T/M298Q-FVII, L305V/V158T/E296V-FVII, L305V/E296V/M298Q-FVII,L305V/V158D/E296V/M298Q-FVII, L305V/V158T/E296V/M298Q-FVII,L305V/V158T/K337A/M298Q-FVII, L305V/V158T/E296V/K337A-FVII,L305V/V158D/K337A/M298Q-FVII, L305V/V158D/E296V/K337A-FVII,L305V/V158D/E296V/M298Q/K337A-FVII, L305V/V158T/E296V/M298Q/K337A-FVII,S314E/K316H-FVII, S314E/K316Q-FVII, S314E/L305V-FVII, S314E/K337A-FVII,S314E/V158D-FVII, S314E/E296V-FVII, S314E/M298Q-FVII, S314E/V158T-FVII,K316H/L305V-FVII, K316H/K337A-FVII, K316H/V158D-FVII, K316H/E296V-FVII,K316H/M298Q-FVII, K316H/V158T-FVII, K316Q/L305V-FVII, K316Q/K337A-FVII,K316Q/V158D-FVII, K316Q/E296V-FVII, K316Q/M298Q-FVII, K316Q/V158T-FVII,S314E/L305V/K337A-FVII, S314E/L305V/V158D-FVII, S314E/L305V/E296V-FVII,S314E/L305V/M298Q-FVII, S314E/L305V/V158T-FVII,S314E/L305V/K337A/V158T-FVII, S314E/L305V/K337A/M298Q-FVII,S314E/L305V/K337A/E296V-FVII, S314E/L305V/K337A/V158D-FVII,S314E/L305V/V158D/M298Q-FVII, S314E/L305V/V158D/E296V-FVII,S314E/L305V/V158T/M298Q-FVII, S314E/L305V/V158T/E296V-FVII,S314E/L305V/E296V/M298Q-FVII, S314E/L305V/V158D/E296V/M298Q-FVII,S314E/L305V/V158T/E296V/M298Q-FVII, S314E/L305V/V158T/K337A/M298Q-FVII,S314E/L305V/V158T/E296V/K337A-FVII, S314E/L305V/V158D/K337A/M298Q-FVII,S314E/L305V/V158D/E296V/K337A-FVII,S314E/L305V/V158D/E296V/M298Q/K337A-FVII,S314E/L305V/V158T/E296V/M298Q/K337A-FVII, K316H/L305V/K337A-FVII,K316H/L305V/V158D-FVII, K316H/L305V/E296V-FVII, K316H/L305V/M298Q-FVII,K316H/L305V/V158T-FVII, K316H/L305V/K337A/V158T-FVII,K316H/L305V/K337A/M298Q-FVII, K316H/L305V/K337A/E296V-FVII,K316H/L305V/K337A/V158D-FVII, K316H/L305V/V158D/M298Q-FVII,K316H/L305V/V158D/E296V-FVII, K316H/L305V/V158T/M298Q-FVII,K316H/L305V/V158T/E296V-FVII, K316H/L305V/E296V/M298Q-FVII,K316H/L305V/V158D/E296V/M298Q-FVII, K316H/L305V/V158T/E296V/M298Q-FVII,K316H/L305V/V158T/K337A/M298Q-FVII, K316H/L305V/V158T/E296V/K337A-FVII,K316H/L305V/V158D/K337A/M298Q-FVII, K316H/L305V/V158D/E296V/K337A-FVII,K316H/L305V/V158D/E296V/M298Q/K337A-FVII,K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/L305V/K337A-FVII,K316Q/L305V/V158D-FVII, K316Q/L305V/E296V-FVII, K316Q/L305V/M298Q-FVII,K316Q/L305V/V158T-FVII, K316Q/L305V/K337A/V158T-FVII,K316Q/L305V/K337A/M298Q-FVII, K316Q/L305V/K337A/E296V-FVII,K316Q/L305V/K337A/V158D-FVII, K316Q/L305V/V158D/M298Q-FVII,K316Q/L305V/V158D/E296V-FVII, K316Q/L305V/V158T/M298Q-FVII,K316Q/L305V/V158T/E296V-FVII, K316Q/L305V/E296V/M298Q-FVII,K316Q/L305V/V158D/E296V/M298Q-FVII, K316Q/L305V/V158T/E296V/M298Q-FVII,K316Q/L305V/V158T/K337A/M298Q-FVII, K316Q/L305V/V158T/E296V/K337A-FVII,K316Q/L305V/V158D/K337A/M298Q-FVII, K316Q/L305V/V158D/E296V/K337A-FVII,K316Q/L305V/V158D/E296V/M298Q/K337A-FVII,K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII,F374Y/V158D-FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII, F374Y/V158T-FVII,F374Y/S314E-FVII, F374Y/L305V-FVII, F374Y/L305V/K337A-FVII,F374Y/L305V/V158D-FVII, F374Y/L305V/E296V-FVII, F374Y/L305V/M298Q-FVII,F374Y/L305V/V158T-FVII, F374Y/L305V/S314E-FVII, F374Y/K337A/S314E-FVII,F374Y/K337A/V158T-FVII, F374Y/K337A/M298Q-FVII, F374Y/K337A/E296V-FVII,F374Y/K337A/V158D-FVII, F374Y/V158D/S314E-FVII, F374Y/V158D/M298Q-FVII,F374Y/V158D/E296V-FVII, F374Y/V158T/S314E-FVII, F374Y/V158T/M298Q-FVII,F374Y/V158T/E296V-FVII, F374Y/E296V/S314E-FVII, F374Y/S314E/M298Q-FVII,F374Y/E296V/M298Q-FVII, F374Y/L305V/K337A/V158D-FVII,F374Y/L305V/K337A/E296V-FVII, F374Y/L305V/K337A/M298Q-FVII,F374Y/L305V/K337A/V158T-FVII, F374Y/L305V/K337A/S314E-FVII,F374Y/L305V/V158D/E296V-FVII, F374Y/L305V/V158D/M298Q-FVII,F374Y/L305V/V158D/S314E-FVII, F374Y/L305V/E296V/M298Q-FVII,F374Y/L305V/E296V/V158T-FVII, F374Y/L305V/E296V/S314E-FVII,F374Y/L305V/M298Q/V158T-FVII, F374Y/L305V/M298Q/S314E-FVII,F374Y/L305V/V158T/S314E-FVII, F374Y/K337A/S314E/V158T-FVII,F374Y/K337A/S314E/M298Q-FVII, F374Y/K337A/S314E/E296V-FVII,F374Y/K337A/S314E/V158D-FVII, F374Y/K337A/V158T/M298Q-FVII,F374Y/K337A/V158T/E296V-FVII, F374Y/K337A/M298Q/E296V-FVII,F374Y/K337A/M298Q/V158D-FVII, F374Y/K337A/E296V/V158D-FVII,F374Y/V158D/S314E/M298Q-FVII, F374Y/V158D/S314E/E296V-FVII,F374Y/V158D/M298Q/E296V-FVII, F374Y/V158T/S314E/E296V-FVII,F374Y/V158T/S314E/M298Q-FVII, F374Y/V158T/M298Q/E296V-FVII,F374Y/E296V/S314E/M298Q-FVII, F374Y/L305V/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/K337A/S314E-FVII, F374Y/E296V/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A-FVII, F374Y/L305V/E296V/M298Q/S314E-FVII,F374Y/V158D/E296V/M298Q/K337A-FVII, F374Y/V158D/E296V/M298Q/S314E-FVII,F374Y/L305V/V158D/K337A/S314E-FVII, F374Y/V158D/M298Q/K337A/S314E-FVII,F374Y/V158D/E296V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q-FVII,F374Y/L305V/V158D/M298Q/K337A-FVII, F374Y/L305V/V158D/E296V/K337A-FVII,F374Y/L305V/V158D/M298Q/S314E-FVII, F374Y/L305V/V158D/E296V/S314E-FVII,F374Y/V158T/E296V/M298Q/K337A-FVII, F374Y/V158T/E296V/M298Q/S314E-FVII,F374Y/L305V/V158T/K337A/S314E-FVII, F374Y/V158T/M298Q/K337A/S314E-FVII,F374Y/V158T/E296V/K337A/S314E-FVII, F374Y/L305V/V158T/E296V/M298Q-FVII,F374Y/L305V/V158T/M298Q/K337A-FVII, F374Y/L305V/V158T/E296V/K337A-FVII,F374Y/L305V/V158T/M298Q/S314E-FVII, F374Y/L305V/V158T/E296V/S314E-FVII,F374Y/E296V/M298Q/K337A/V158T/S314E-FVII,F374Y/V158D/E296V/M298Q/K337A/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/S314E-FVII,F374Y/L305V/E296V/M298Q/V158T/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A/V158T-FVII,F374Y/L305V/E296V/K337A/V158T/S314E-FVII,F374Y/L305V/M298Q/K337A/V158T/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/K337A-FVII,F374Y/L305V/V158D/E296V/K337A/S314E-FVII,F374Y/L305V/V158D/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII, S52A-Factor VII,S60A-Factor VII; R152E-Factor VII, S344A-Factor VII, T106N-FVII,K143N/N145T-FVII, V253N-FVII, R290N/A292T-FVII, G291N-FVII,R315N/V317T-FVII, K143N/N145T/R315N/V317T-FVII; and FVII havingsubstitutions, additions or deletions in the amino acid sequence from233Thr to 240Asn; FVII having substitutions, additions or deletions inthe amino acid sequence from 304Arg to 329Cys; and FVII havingsubstitutions, additions or deletions in the amino acid sequence from153Ile to 223Arg.

Factor VII variants having substantially the same or improved biologicalactivity relative to wild-type Factor VIIa encompass those that exhibitat least about 25%, such as, e.g., at least about 50%, at least about75%, at least about 90%, at least about 120, at least about 130, or atleast about 150% of the specific activity of wild-type Factor VIIa thathas been produced in the same cell type, when tested in one or more of aclotting assay, proteolysis assay, or TF binding assay as describedabove. Factor VII variants having substantially reduced biologicalactivity relative to wild-type Factor VIIa are those that exhibit lessthan about 25%, preferably less than about 10%, more preferably lessthan about 5% and most preferably less than about 1% of the specificactivity of wild-type Factor VIIa that has been produced in the samecell type when tested in one or more of a clotting assay, proteolysisassay, or TF binding assay as described below. Factor VII variantshaving a substantially modified biological activity relative towild-type Factor VII include, without limitation, Factor VII variantsthat exhibit TF-independent Factor X proteolytic activity and those thatbind TF but do not cleave Factor X.

The biological activity of Factor VIIa in blood clotting derives fromits ability to (i) bind to tissue factor (TF) and (ii) catalyze theproteolytic cleavage of Factor IX or Factor X to produce activatedFactor IX or X (Factor IXa or Xa, respectively). For purposes of theinvention, Factor VIIa biological activity may be quantified bymeasuring the ability of a preparation to promote blood clotting usingFactor VII-deficient plasma and thromboplastin, as described, e.g., inU.S. Pat. No. 5,997,864. In this assay, biological activity is expressedas the reduction in clotting time relative to a control sample and isconverted to “Factor VII units” by comparison with a pooled human serumstandard containing 1 unit/ml Factor VII activity. Alternatively, FactorVIIa biological activity may be quantified by (i) measuring the abilityof Factor VIIa to produce of Factor Xa in a system comprising TFembedded in a lipid membrane and Factor X. (Persson et al., J. Biol.Chem. 272:19919-19924, 1997); (ii) measuring Factor X hydrolysis in anaqueous system (see, “General Methods” below); (iii) measuring itsphysical binding to TF using an instrument based on surface plasmonresonance (Persson, FEBS Letts. 413:359-363, 1997) (iv) measuringhydrolysis of a synthetic substrate (see, “General Methods” below); and(v) measuring generation of thrombin in a TF-independent in vitro system(see, “General Methods” below).

Factor IX Polypeptides and Factor IX-Related Polypeptides

The present invention encompasses factor IX polypeptides, such as, e.g.,those having the amino acid sequence disclosed in, e.g., Jaye et al.,Nucleic Acids Res. 11: 2325-2335, 1983. (wild-type human factor IX).

In practicing the present invention, any factor IX polypeptide may beused that is effective in preventing or treating bleeding. This includesfactor IX polypeptides derived from blood or plasma, or produced byrecombinant means.

As used herein, “factor IX polypeptide” encompasses, without limitation,factor IX, as well as factor IX-related polypeptides. The term “factorIX” is intended to encompass, without limitation, polypeptides havingthe amino acid sequence as described in Jaye et al., Nucleic Acids Res.1983 (see above) (wild-type human factor IX), as well as wild-typeFactor IX derived from other species, such as, e.g., bovine, porcine,canine, murine, and salmon Factor IX. It further encompasses naturalallelic variations of Factor IX that may exist and occur from oneindividual to another. Also, degree and location of glycosylation orother post-translation modifications may vary depending on the chosenhost cells and the nature of the host cellular environment. The term“Factor IX” is also intended to encompass Factor IX polypeptides intheir uncleaved (zymogen) form, as well as those that have beenproteolytically processed to yield their respective bioactive forms,which may be designated Factor IXa.

“Factor IX-related polypeptides” include, without limitation, factor IXpolypeptides that have either been chemically modified relative to humanfactor IX and/or contain one or more amino acid sequence alterationsrelative to human factor IX (i.e., factor IX variants), and/or containtruncated amino acid sequences relative to human factor IX (i.e., factorIX fragments). Such factor IX-related polypeptides may exhibit differentproperties relative to human factor IX, including stability,phospholipid binding, altered specific activity, and the like.

The term “factor IX-related polypeptides” are intended to encompass suchpolypeptides in their uncleaved (zymogen) form, as well as those thathave been proteolytically processed to yield their respective bioactiveforms, which may be designated “factor IXa-related polypeptides” or“activated factor IX-related polypeptides”.

As used herein, “factor IX-related polypeptides” encompasses, withoutlimitation, polypeptides exhibiting substantially the same or improvedbiological activity relative to wild-type human factor IX, as well aspolypeptides, in which the factor IX biological activity has beensubstantially modified or reduced relative to the activity of wild-typehuman factor IX. These polypeptides include, without limitation, factorIX or factor IXa that has been chemically modified and factor IXvariants into which specific amino acid sequence alterations have beenintroduced that modify or disrupt the bioactivity of the polypeptide.

It further encompasses polypeptides with a slightly modified amino acidsequence, for instance, polypeptides having a modified N-terminal endincluding N-terminal amino acid deletions or additions, and/orpolypeptides that have been chemically modified relative to human factorIX.

Factor IX-related polypeptides, including variants of factor IX, whetherexhibiting substantially the same or better bioactivity than wild-typefactor IX, or, alternatively, exhibiting substantially modified orreduced bioactivity relative to wild-type factor IX, include, withoutlimitation, polypeptides having an amino acid sequence that differs fromthe sequence of wild-type factor IX by insertion, deletion, orsubstitution of one or more amino acids.

Factor IX-related polypeptides, including variants, encompass those thatexhibit at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 100%, atleast about 110%, at least about 120%, and at least about 130%, of thespecific activity of wild-type factor IX that has been produced in thesame cell type, when tested in the factor IX activity assay as describedin the present specification.

Factor IX-related polypeptides, including variants, having substantiallythe same or improved biological activity relative to wild-type factor IXencompass those that exhibit at least about 25%, preferably at leastabout 50%, more preferably at least about 75%, more preferably at leastabout 100%, more preferably at least about 110%, more preferably atleast about 120%, and most preferably at least about 130% of thespecific biological activity of wild-type human factor IX that has beenproduced in the same cell type when tested in one or more of thespecific factor IX activity assays as described. For purposes of theinvention, factor IX biological activity may be quantified as describedlater in the present description (see “General Methods”).

Factor IX-related polypeptides, including variants, having substantiallyreduced biological activity relative to wild-type factor IX are thosethat exhibit less than about 25%, preferably less than about 10%, morepreferably less than about 5% and most preferably less than about 1% ofthe specific activity of wild-type factor IX that has been produced inthe same cell type when tested in one or more of the specific factor IXactivity assays as described above.

Non-limiting examples of factor IX polypeptides include plasma-derivedhuman factor IX as described, e.g., in Chandra et al., Biochem. Biophys.Acta 1973, 328:456; Andersson et al., Thromb. Res. 1975, 7:451; Suomelaet al., Eur. J. Biochem. 1976, 71:145.

Suitable assays for testing for factor IX activity, and therebyproviding means for selecting suitable factor IX variants for use in thepresent invention, can be performed as simple in vitro tests asdescribed, for example, in Wagenvoord et al., Haemostasis 1990;20(5):276-88. Factor IX biological activity may also be quantified bymeasuring the ability of a preparation to correct the clotting time offactor IX-deficient plasma, e.g., as described in Nilsson et al., 1959.(Nilsson I M, Blombaeck M, Thilen A, von Francken I., Carriers ofhaemophilia A—A laboratory study, Acta Med Scan 1959; 165:357). In thisassay, biological activity is expressed as units/ml plasma (1 unitcorresponds to the amount of FIX present in normal pooled plasma.

In some embodiments of the invention, the factor IX are factorIX-related polypeptides wherein the ratio between the activity of saidfactor IX polypeptide and the activity of native human factor IX(wild-type factor IX) is at least about 1.25 when tested in the“chromogenic assay” (see below); in other embodiments, the ratio is atleast about 2.0; in further embodiments, the ratio is at least about4.0.

O-Linked Glycosylation

In practicing the present invention, the pattern of oligosaccharides maybe determined using any method known in the art, including, withoutlimitation: high-performance liquid chromatography (HPLC); capillaryelectrophoresis (CE); nuclear magnetic resonance (NMR); massspectrometry (MS) using ionization techniques such as fast-atombombardment, electrospray, or matrix-assisted laser desorption (MALDI);gas chromatography (GC); and treatment with exoglycosidases inconjunction with anion-exchange (AIE)-HPLC, size-exclusionchromatography (SEC), or MS. See, e.g., Weber et al., Anal. Biochem.225:135 (1995); Klausen et al., J. Chromatog. 718:195 (1995); Morris etal., in Mass Spectrometry of Biological Materials, McEwen et al., eds.,Marcel Dekker, (1990), pp 137-167; Conboy et al., Biol. Mass Spectrom.21:397, 1992; Hellerqvist, Meth. Enzymol. 193:554 (1990); Sutton et al.,Anal. Biochem. 318:34 (1994); Harvey et al., Organic Mass Spectrometry29:752 (1994).

The relative content of O-glycoforms can be determined, for example, bytryptic peptide mapping. In short, the glycoprotein is digested withtrypsin and the polypeptides containing the O-glycosylation site areseparated according to the glycan structure by RP-HPLC chromatography,mass spectrometry or another suitable analytical separation technique.If necessary in order to obtaining a suitable separation, theglycoprotein can prior to the digestion with trypsin be reduced andalkylated and the polypeptide chain containing the O-glycosylation siteis purified by, e.g. RP-HPLC chromatography. Then the purifiedpolypeptide is subjected to tryptic digestion followed by analysis asdescribed above.

Methods for Producing Glycoprotein Preparations Having a PredeterminedPattern of O-Linked Oligosaccharides

The origin of the acceptor glycoprotein is not a critical aspect of theinvention. Typically, the glycoprotein will be expressed in a culturedprokaryote cell or eukaryote cell such as a mammalian, yeast insect,fungal or plant cell. The protein, however, may also be isolated from anatural source such as plasma, serum or blood. The glycoprotein caneither be a full length protein or a fragment.

The invention provides compositions that include glycoprotein speciesthat have a substantially uniform glycosylation pattern. The methods areuseful for remodelling or altering the glycosylation pattern present ona glycoprotein upon its initial expression. Thus, the methods of theinvention provide a practical means for large-scale preparation ofglycoforms having pre-selected or pre-determined uniform derivatizationpatterns. The methods are particularly well suited for modification oftherapeutic peptides, including but not limited to, glycoproteins thatare incompletely glycosylated during production in cell culture cells ortransgenic animals. However, the preparations and compositions of theinvention may also be prepared by purification of natural sources, suchas plasma, serum or blood, or cell culture fluids and isolating thedesired glycoforms therefrom.

The polypeptides to be re-modelled in accordance with the invention aretypically prepared by cell culture processes. Suitable host cellsinclude, without limitation, human cells expressing an endogenous genesuch as, e.g., a Factor VII, IX, X, or XII gene or a protein Z gene. Inthese cells, the endogenous gene may be intact or may have been modifiedin situ, or a sequence outside the endogenous gene may have beenmodified in situ to alter the expression of the endogenous glycoproteingene. Any human cell capable of expressing an endogenous glycoproteingene may be used. Other, included host cells are heterologous host cellsprogrammed to express a glycoprotein such as, e.g., human Factor VII orIX or X or XII from a recombinant gene. The host cells may bevertebrate, insect, or fungal cells. Preferably, the cells are mammaliancells capable of the entire spectrum of mammalian N-linkedglycosylation; O-linked glycosylation; and γ-carboxylation. See, e.g.,U.S. Pat. Nos. 4,784,950. Preferred mammalian cell lines include the CHO(ATCC CCL 61), COS-1 (ATCC CRL 1650), baby hamster kidney (BHK) andHEK293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977)cell lines. A preferred BHK cell line is the tk⁻ ts13 BHK cell line(Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982),hereinafter referred to as BHK 570 cells. The BHK 570 cell line isavailable from the American Type Culture Collection, 12301 Parklawn Dr.,Rockville, Md. 20852, under ATCC accession number CRL 10314. A tk⁻ ts13BHK cell line is also available from the ATCC under accession number CRL1632. In addition, a number of other cell lines may be used, includingRat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCCCRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469(ATCC CCL 9.1) and DUKX cells (CHO cell line) (Urlaub and Chasin, Proc.Natl. Acad. Sci. USA 77:4216-4220, 1980). (DUKX cells also referred toas CXB11 cells), and DG44 (CHO cell line) (Cell, 33:405, 1983, andSomatic Cell and Molecular Genetics 12:555, 1986). Also useful are 3T3cells, Namalwa cells, myelomas and fusions of myelomas with other cells.Suitable host cells include BHK 21 cells that have been adapted to growin the absence of serum and have been programmed to express Factor VII.The cells may be mutant or recombinant cells that express aqualitatively or quantitatively different spectrum of glycosylationenzymes (such as, e.g., glycosyl transferases and/or glycosidases) thanthe cell type from which they were derived. The cells may also beprogrammed to express other heterologous peptides or proteins,including, e.g., truncated forms of Factor VII. The host cells may alsobe CHO cells that have been programmed to co-express both the Factor VIIpolypeptide of interest (i.e., Factor VII or a Factor-VII-relatedpolypeptide) and another heterologous peptide or polypeptide such as,e.g., a modifying enzyme or a Factor VII fragment.

Methods: The present invention encompasses methods for producing apreparation comprising a predetermined serine/threonine-linked glycoformpattern as described above and, in further embodiments, methods foroptimizing the glycoform distribution of a glycoprotein (see FIG. 3).The individual process steps described can be applied in differentcombinations in order to obtain the desired glycoform pattern.Non-limiting examples are given below.

In one aspect, these methods are carried out by the steps of:

(a) obtaining a preparation of a glycoprotein containing aCys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine formspart of a Glc-O-Ser/Thr covalent bond from a cell in which it isprepared; e.g., from an engineered cell (cell culture) or by isolatingthe glycoprotein from a natural source;

(b) contacting the glycoprotein preparation with an activated donor ofthe desired mono- or oligosaccharide moiety and an enzyme suitable fortransferring the desired mono- or oligo-saccharide group underconditions appropriate for transferring the mono- or oligo-saccharidegroup from the donor moiety to the acceptor moiety, thereby producingthe glycopeptide having an altered glycosylation pattern.

In another aspect, these methods are carried out by the steps of:

(aa) obtaining a preparation of a glycoprotein containing aCys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine formspart of a Glc-O-Ser/Thr covalent bond from a cell in which it isprepared; e.g., from an engineered cell (cell culture) or by isolatingthe glycoprotein from a natural source;

(bb) contacting the glycoprotein preparation with an enzyme suitable forremoving the terminal mono- or oligo-saccharide group under conditionsappropriate for removing said mono- or oligo-saccharide group, therebyproducing the glycopeptide having an altered glycosylation pattern.

In one embodiment, the methods comprise a combination of steps (b) and(bb). In one embodiment the methods further comprise a step of isolatingthe glycoprotein having an altered glycosylation pattern.

In one embodiment, the methods comprise a further step of:

Analyzing the structure of the oligosaccharides linked to thepolypeptides to determine a glycoform pattern, and, optionally,repeating steps (b) and/or (bb) until a desired glycoform pattern isachieved.

These methods may further comprise the step of subjecting preparationshaving predetermined glycoform patterns to at least one test ofbioactivity (including, e.g., clotting, Factor X proteolysis, or TFbinding) or other functionality (such as, e.g., pharmacokinetic profileor stability), and correlating particular glycoform patterns withparticular bioactivity or functionality profiles in order to identify adesired glycoform pattern.

In one embodiment, the desired glycoform pattern is a substantiallyuniform glucose-O-serine/threonine glycosylation: In this embodiment,wherein the initially obtained glycoprotein contains terminal xylose themethod (METHOD B) comprises the steps of:

(a) obtaining a preparation of a glycoprotein containing aCys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine formspart of a Glc-O-Ser/Thr covalent bond from a cell in which it isprepared; e.g., from an engineered cell (cell culture) or by isolatingthe glycoprotein from a natural source;

(b) contacting the preparation obtained in step (a) with a xylosidaseunder conditions appropriate for removing xylose residues from theglycoprotein, thereby producing the glycoprotein having an alteredglycosylation pattern.

In one embodiment, the method further includes the step of isolating theglycoprotein prepared in step b having a Glc-O-Ser/Thr glycosylation.

In one embodiment, the method further includes the step of analysing thestructure of the oligosaccharides linked to the polypeptides todetermine a glycoform pattern, and, optionally, repeating step (b) untilthe desired glycoform pattern is achieved.

In another embodiment for making a desired glycoforms pattern in theform of a substantially uniform glucose-O-serine/threonineglycosylation, the method (METHOD C) comprises the steps of:

(a) obtaining a preparation of a polypeptide containing aCys-X1-Ser/Thr-X2-Pro-Cys motif, e.g., from an engineered cell (cellculture) or by isolating the glycoprotein from a natural source;

(b) contacting the preparation obtained in step (a) with aO-glucosyltransferase and an activated glucose donor under conditionsappropriate for transferring a glucose residue from the glucose donormoiety to the serine/threonine acceptor moiety, thereby producing thepolypeptide having an altered glycosylation pattern.

In one embodiment, the method further includes the step of isolating theglycoprotein prepared in step b having a Glc-O-Ser/Thr glycosylation.

In one embodiment, the method further includes the step of analysing thestructure of the oligosaccharides linked to the polypeptides todetermine a glycoform pattern, and, optionally, repeating step (b) untilthe desired glycoform pattern is achieved.

In one embodiment, the desired glycoform pattern is a substantiallyuniform xylose-glucose-O-serine/threonine glycosylation: In thisembodiment, the method (METHOD A1) comprises the steps of:

(a) obtaining a preparation of a glycoprotein containing aCys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine formspart of a Glc-O-Ser/Thr covalent bond; e.g., from an engineered cell(cell culture) or by isolating the glycoprotein from a natural source;

(b) contacting the preparation obtained in step (a) with UDP-D-xylose:β-D-glucoside α-1,3-D-xylosyltransferase and an activated xylosyl donorunder conditions appropriate for transferring a xylose residue from thexylose donor moiety to the acceptor moiety, thereby producing theglycopeptide having an altered glycosylation pattern.

In one embodiment, the method further includes the step of isolating theglycoprotein prepared in step b having a Xyl-Glc-O-Ser/Thrglycosylation.

In one embodiment, the method further includes the step of analysing thestructure of the oligosaccharides linked to the polypeptides todetermine a glycoform pattern, and, optionally, repeating step (b) untilthe desired glycoform pattern is achieved.

In one embodiment, the method further includes the step of removingterminal xylose-residues by subjecting the preparation obtained in step(a) to METHOD B prior to step (b).

In one embodiment, the desired glycoform pattern is a substantiallyuniform xylose-xylose-glucose-O-serine/threonine glycosylation: In thisembodiment, the method (METHOD A2) comprises the steps of:

(a) obtaining a preparation of a glycoprotein containing aCys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine formspart of a Glc-O-Ser/Thr covalent bond; e.g., from an engineered cell(cell culture) or by isolating the glycoprotein from a natural source;

(b) contacting the preparation obtained in step (a) with UDP-D-xylose:β-D-glucoside α-1,3-D-xylosyltransferase and an activated xylosyl donorunder conditions appropriate for transferring a xylose residue from thexylose donor moiety to the acceptor moiety, thereby producing theglycopeptide having an altered glycosylation pattern.

(c) contacting the preparation obtained in step (b) with UDP-D-xylose:α-D-xyloside α-1,3-xylosyltransferase and an activated xylosyl donorunder conditions appropriate for transferring a xylose residue from thexylose donor moiety to the acceptor moiety, thereby producing theglycopeptide having an altered glycosylation pattern.

In one embodiment, the method further includes the step of isolating thepreparation obtained in step (b) prior to subjecting the preparation tostep (c).

In one embodiment, the method further includes the step of isolating theglycoprotein prepared in step (c) having a Xyl-Xyl-Glc-O-Ser/Thrglycosylation.

In one embodiment, the method further includes the step of analysing thestructure of the oligosaccharides linked to the polypeptides todetermine a glycoform pattern, and, optionally, repeating step (b)and/or step (c) until the desired glycoform pattern is achieved.

In one embodiment, the method further includes the step of removingterminal xylose-residues by subjecting the preparation obtained in step(a) to METHOD B prior to step (b).

In different embodiments, the glycoprotein exhibits substantiallyuniform Xyl-Xyl-Glc-O-Ser glycosylation, Xyl-Glc-O-Ser glycosylation,and Glc-O-Ser glycosylation; Ser being the serine of the containedCys-X1-Ser-X2-Pro-Cys motif (X1 and X2 independently being any aminoacid residue). In other, different embodiments, the glycoproteinexhibits substantially uniform Xyl-Xyl-Glc-O-Thr glycosylation,Xyl-Glc-O-Thr glycosylation, and Glc-O-Thr glycosylation; Thr being thethreonine of the contained Cys-X1-Thr-X2-Pro-Cys motif (X1 and X2independently being any amino acid residue).

In different embodiments, the polypeptides are selected from the listof: Factor VII polypeptides, Factor VII-related polypeptides, Factor IXpolypeptides, Factor IX-related polypeptides, Factor X polypeptides, andFactor X-related polypeptides.

In preferred embodiments, the glycoprotein preparation is selected fromthe list of:

Factor VII polypeptides exhibiting substantially uniformXyl-Xyl-Glc-O-Ser52 glycosylation,Factor VII polypeptides exhibiting substantially uniform Xyl-Glc-O-Ser52glycosylationFactor VII polypeptides exhibiting substantially uniform Glc-O-Ser52glycosylationFactor VII-related polypeptides exhibiting substantially uniformXyl-Xyl-Glc-O-Ser52 glycosylationFactor VII-related polypeptides exhibiting substantially uniformXyl-Glc-O-Ser52 glycosylationFactor VII-related polypeptides exhibiting substantially uniformGlc-O-Ser52 glycosylationFactor VII variants exhibiting substantially uniform Xyl-Xyl-Glc-O-Ser52glycosylationFactor VII variants exhibiting substantially uniform Xyl-Glc-O-Ser52glycosylationFactor VII variants exhibiting substantially uniform Glc-O-Ser52glycosylationFactor IX polypeptides exhibiting substantially uniformXyl-Xyl-Glc-O-Ser53 glycosylationFactor IX polypeptides exhibiting substantially uniform Xyl-Glc-O-Ser53glycosylationFactor IX polypeptides exhibiting substantially uniform Glc-O-Ser53glycosylationFactor IX-related polypeptides exhibiting substantially uniformXyl-Xyl-Glc-O-Ser53 glycosylationFactor IX-related polypeptides exhibiting substantially uniformXyl-Glc-O-Ser53 glycosylationFactor IX-related polypeptides exhibiting substantially uniformGlc-O-Ser53 glycosylationFactor IX variants exhibiting substantially uniform Xyl-Xyl-Glc-O-Ser53glycosylationFactor IX variants exhibiting substantially uniform Xyl-Glc-O-Ser53glycosylationFactor IX variants exhibiting substantially uniform Glc-O-Ser53glycosylation

It is to be understood that oligosaccharides such as Xyl-Xyl- may alsobe transferred to the acceptor Glc-O-Ser/Thr moiety by using a suitabletransferring enzyme and an activated Xyl-Xyl- donor.

Chromatographic method: The present invention also encompasseshydrophobic interaction chromatographic methods for producing apreparation comprising a predetermined serine/threonine-linked glycoformpattern as described above, and for purifying a O-glycosylatedpolypeptide having a desired glycoform pattern from a compositioncomprising said polypeptide and polypeptides having unwanted glycoformpatterns.

In one aspect, the method comprises the following steps:

(a) obtaining a preparation of a glycoprotein containing aCys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine formspart of a Glc-O-Ser/Thr covalent bond from a cell in which it isprepared; e.g., from an engineered cell (cell culture) or by isolatingthe glycoprotein from a natural source;

(b) binding the glycoprotein to an hydrophobic interaction materialusing a solution comprising water, optionally a salt component, andoptionally a buffer,

(c) optionally washing the hydrophobic interaction material using asolution comprising water, optionally a salt component, and optionally abuffer so as to elute contaminants from the hydrophobic interactionmaterial;

(d) washing the hydrophobic interaction material using a solutioncomprising an organic modifier, water, optionally a salt component, andoptionally a buffer, at a linear or step gradient or isocratically insalt component so as to separate glycoproteins having a desiredglycoform patter from glycoproteins not having the desired glycoformfrom the hydrophobic interaction material;

(e) collecting the fraction containing the glycoproteins having thedesired glycoform pattern.

In one embodiment, the above-described methods further includes the stepof repeating steps (a) to e) by subjecting the preparation obtained instep (e) to steps (a) to (e). This further step may be repeated morethan once if deemed necessary.

It is to be understood that the preparations according to the inventionmay also be prepared by a process comprising a combination ofpurification steps whereby glycoprotein species having the desiredglycosylation are captured from the cell culture liquid or naturalsource of origin and the above-described enzymatic methods.

The above-described methods may further comprise the step of subjectingpreparations having predetermined glycoform patterns to at least onetest of bioactivity (including, e.g., clotting, Factor X proteolysis, orTF binding) or other functionality (such as, e.g., pharmacokineticprofile or stability), and correlating particular glycoform patternswith particular bioactivity or functionality profiles in order toidentify a desired glycoform pattern.

Further enzymatic treatments may be used in connection with the abovemethods to modify the oligosaccharide pattern of N- or O-linked glycansof a preparation; such treatments include, without limitation, treatmentwith one or more of sialidase (neuraminidase), galactosidase,fucosidase; galactosyl transferase, fucosyl transferase, and/orsialyltransferase, in a sequence and under conditions that achieve adesired modification in the distribution of oligosaccharide chainshaving particular terminal structures. Glycosyl transferases arecommercially available from Calbiochem (La Jolla, Calif.) andglycosidases are commercially available from Glyko, Inc., (Novato,Calif.).

Glycoprotein Preparations

As used herein, a “glycoprotein preparation” refers to a plurality ofglycoforms that have been separated from the cell in which they weresynthesized. The glycoprotein preparation include inactivated forms,activated forms, functionally related polypeptides such as, e.g.,variants and chemically modified forms, that have been separated fromthe cell in which they were synthesized.

For example, as used herein, a “Factor VII preparation” refers to aplurality of Factor VII polypeptides, Factor VIIa polypeptides, orFactor VII-related polypeptides, including variants and chemicallymodified forms, that have been separated from the cell in which theywere synthesized or isolated from a natural source. Likewise, a “FactorIX preparation” refers to a plurality of Factor IX polypeptides, FactorIXa polypeptides, or Factor IX-related polypeptides, including variantsor chemically modified forms, that have been separated from the cell inwhich they were synthesized or isolated from a natural source (e.g.,plasma, serum, blood).

Separation of polypeptides from their cell of origin may be achieved byany method known in the art, including, without limitation, removal ofcell culture medium containing the desired product from an adherent cellculture; centrifugation or filtration to remove non-adherent cells; andthe like.

Optionally, the polypeptides may be further purified. Purification maybe achieved using any method known in the art, including, withoutlimitation, affinity chromatography, such as, e.g., on an anti-FactorVII or anti-Factor IX antibody column (see, e.g., Wakabayashi et al., J.Biol. Chem. 261:11097, 1986; and Thim et al., Biochem. 27:7785, 1988);hydrophobic interaction chromatography; ion-exchange chromatography;size exclusion chromatography; electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction and the like. See,generally, Scopes, Protein Purification, Springer-Verlag, New York,1982; and Protein Purification, J.-C. Janson and Lars Ryden, editors,VCH Publishers, New York, 1989. Following purification, the preparationpreferably contains less than about 10% by weight, more preferably lessthan about 5% and most preferably less than about 1%, of non-relatedproteins derived from the host cell.

Factor VII and Factor VII-related polypeptides, Factor IX and FactorIX-related polypeptides, or Factor X and Factor X-related polypeptides,respectively, may be activated by proteolytic cleavage, using FactorXIIa or other proteases having trypsin-like specificity, such as, e.g.,Factor IXa, kallikrein, Factor Xa, and thrombin. See, e.g., Osterud etal., Biochem. 11:2853 (1972); Thomas, U.S. Pat. No. 4,456,591; andHedner et al., J. Clin. Invest. 71:1836 (1983). Alternatively, FactorVII, IX or X, respectively, may be activated by passing it through anion-exchange chromatography column, such as Mono Q® (Pharmacia) or thelike. The resulting activated polypeptide, e.g., Factor VII, may then beformulated and administered as described below.

Functional Properties of Glycoprotein Preparations

The preparations of glycoproteins having predetermined oligosaccharidepatterns according to the invention (including Factor VII polypeptides,Factor VII-related polypeptides, Factor IX polypeptides and FactorIX-related polypeptides) exhibit improved functional properties relativeto reference preparations. The improved functional properties mayinclude, without limitation, a) physical properties such as, e.g.,storage stability; b) pharmacokinetic properties such as, e.g.,bioavailability and half-life; c) immunogenicity in humans, and d)biological activity, such as, e.g., clotting activity.

A reference preparation refers to a preparation comprising a polypeptidethat is identical to that contained in the preparation of the inventionto which it is being compared (such as, e.g., wild-type Factor VII orwild-type Factor IX or a particular variant or chemically modified form)except for exhibiting a different pattern of serine/threonine-linkedglycosylation.

Storage stability of a glycoprotein (e.g., Factor VII) preparation maybe assessed by measuring (a) the time required for 20% of thebioactivity of a preparation to decay when stored as a dry powder at 25°C. and/or (b) the time required for a doubling in the proportion of(e.g., Factor VIIa) aggregates of said glycoprotein in the preparation.

In some embodiments, the preparations of the invention exhibit anincrease of at least about 30%, preferably at least about 60% and morepreferably at least about 100%, in the time required for 20% of thebioactivity to decay relative to the time required for the samephenomenon in a reference preparation, when both preparations are storedas dry powders at 25° C. Bioactivity measurements may be performed usingany of a clotting assay, proteolysis assay, TF-binding assay, orTF-independent thrombin generation assay.

In some embodiments, the preparations of the invention exhibit anincrease of at least about 30%, preferably at least about 60%, and morepreferably at least about 100%, in the time required for doubling ofaggregates relative to a reference preparation, when both preparationsare stored as dry powders at 25° C. The contents of aggregates may bedetermined according to methods known to the skilled person, such as,e.g., gel permeation HPLC methods. For example, the content of FactorVII aggregates is determined by gel permeation HPLC on a Protein Pak 300SW column (7.5×300 mm) (Waters, 80013) as follows. The column isequilibrated with Eluent A (0.2 M ammonium sulfate, 5% isopropanol, pHadjusted to 2.5 with phosphoric acid, and thereafter pH is adjusted to7.0 with triethylamine), after which 25 μg of sample is applied to thecolumn. Elution is with Eluent A at a flow rate of 0.5 ml/min for 30min, and detection is achieved by measuring absorbance at 215 nm. Thecontent of aggregates is calculated as the peak area of the Factor VIIaggregates/total area of Factor VII peaks (monomer and aggregates).

“Bioavailability” refers to the proportion of an administered dose of a(e.g., Factor VII or Factor VII-related) glycoprotein preparation thatcan be detected in plasma at predetermined times after administration.Typically, bioavailability is measured in test animals by administeringa dose of between about 25-250 μg/kg of the preparation; obtainingplasma samples at predetermined times after administration; anddetermining the content of (e.g., Factor VII or Factor VII-related)glycosylated polypeptides in the samples using one or more of a clottingassay (or any bioassay), an immunoassay, or an equivalent. The data aretypically displayed graphically as polypeptide [e.g., Factor VII] v.time and the bioavailability is expressed as the area under the curve(AUC). Relative bioavailability of a test preparation refers to theratio between the AUC of the test preparation and that of the referencepreparation.

In some embodiments, the preparations of the present invention exhibit arelative bioavailability of at least about 110%, preferably at leastabout 120%, more preferably at least about 130% and most preferably atleast about 140% of the bioavailability of a reference preparation. Thebioavailability may be measured in any mammalian species, preferablydogs, and the predetermined times used for calculating AUC may encompassdifferent increments from 10 min-8 h.

“Half-life” refers to the time required for the plasma concentration of(e.g., Factor VII polypeptides of Factor VII-related polypeptides) theglycoprotein to decrease from a particular value to half of that value.Half-life may be determined using the same procedure as forbioavailability. In some embodiments, the preparations of the presentinvention exhibit an increase in half-life of at least about 0.25 h,preferably at least about 0.5 h, more preferably at least about 1 h, andmost preferably at least about 2 h, relative to the half-life of areference preparation.

“Immunogenicity” of a preparation refers to the ability of thepreparation, when administered to a human, to elicit a deleteriousimmune response, whether humoral, cellular, or both. Factor VIIapolypeptides and Factor VIIa-related polypeptides are not known toelicit detectable immune responses in humans. Nonetheless, in any humansub-population, there may exist individuals who exhibit sensitivity toparticular administered proteins. Immunogenicity may be measured byquantifying the presence of anti-Factor VII antibodies and/or FactorVII-responsive T-cells in a sensitive individual, using conventionalmethods known in the art. In some embodiments, the preparations of thepresent invention exhibit a decrease in immunogenicity in a sensitiveindividual of at least about 10%, preferably at least about 25%, morepreferably at least about 40% and most preferably at least about 50%,relative to the immunogenicity for that individual of a referencepreparation.

Pharmaceutical compositions and Methods of Use

The preparations of the present invention may be used to treat anysyndrome responsive to the relevant glycoprotein. Factor VII-, FIX andFX-responsive syndromes, respectively, include syndromes such as, e.g.,bleeding disorders, including, without limitation, those caused byclotting factor deficiencies (e.g., haemophilia A and B or deficiency ofcoagulation factors XI or VII); by thrombocytopenia or von Willebrand'sdisease, or by clotting factor inhibitors, or excessive bleeding fromany cause. The preparations may also be administered to patients inassociation with surgery or other trauma or to patients receivinganticoagulant therapy.

Preparations comprising Factor VII-related polypeptides according to theinvention, which have substantially reduced bioactivity relative towild-type Factor VII, may be used as anticoagulants, such as, e.g., inpatients undergoing angioplasty or other surgical procedures that mayincrease the risk of thrombosis or occlusion of blood vessels as occurs,e.g., in restenosis. Other medical indications for which anticoagulantsare pre-scribed include, without limitation, deep vein thrombosis,pulmonary embolism, stroke, disseminated intravascular coagulation(DIC), fibrin deposition in lungs and kidneys associated withgram-negative endotoxemia, myocardial infarction; Acute RespiratoryDistress Syndrome (ARDS), Systemic Inflammatory Response Syndrome(SIRS), Hemolytic Uremic Syndrome (HUS), MOF, and TTP.

Pharmaceutical compositions comprising the Factor VII and FactorVII-related preparations according to the present are primarily intendedfor parenteral administration for prophylactic and/or therapeutictreatment. Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intravenously, subcutaneously, or intramuscularly.They may be administered by continuous or pulsatile infusion.

Pharmaceutical compositions or formulations comprise a preparationaccording to the invention in combination with, preferably dissolved in,a pharmaceutically acceptable carrier, preferably an aqueous carrier ordiluent. A variety of aqueous carriers may be used, such as water,buffered water, 0.4% saline, 0.3% glycine and the like. The preparationsof the invention can also be formulated into liposome preparations fordelivery or targeting to the sites of injury. Liposome preparations aregenerally described in, e.g., U.S. Pat. Nos. 4,837,028, 4,501,728, and4,975,282. The compositions may be sterilised by conventional,well-known sterilisation techniques. The resulting aqueous solutions maybe packaged for use or filtered under aseptic conditions andlyophilised, the lyophilised preparation being combined with a sterileaqueous solution prior to administration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances or adjuvants, including, without limitation, pH adjusting andbuffering agents and/or tonicity adjusting agents, such as, for example,sodium acetate, sodium lactate, sodium chloride, potassium chloride,calcium chloride, etc.

The concentration of Factor VII or Factor VII-related polypeptides inthese formulations can vary widely, i.e., from less than about 0.5% byweight, usually at or at least about 1% by weight to as much as 15 or20% by weight and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected.

Thus, a typical pharmaceutical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution and 10mg of the preparation. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in, for example, Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa.(1990).

The compositions containing the preparations of the present inventioncan be administered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered to a subjectalready suffering from a disease, as described above, in an amountsufficient to cure, alleviate or partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as“therapeutically effective amount”. Effective amounts for each purposewill depend on the severity of the disease or injury as well as theweight and general state of the subject. In general, however, theeffective amount will range from about 0.05 mg up to about 500 mg of thepreparation per day for a 70 kg subject, with dosages of from about 1.0mg to about 200 mg of the preparation per day being more commonly used.It will be understood that determining an appropriate dosage may beachieved using routine experimentation, by constructing a matrix ofvalues and testing different points in the matrix.

Local delivery of the preparations of the present invention, such as,for example, topical application, may be carried out, e.g., by means ofa spray, perfusion, double balloon catheters, stents, incorporated intovascular grafts or stents, hydrogels used to coat balloon catheters, orother well established methods. In any event, the pharmaceuticalcompositions should provide a quantity of the preparation sufficient toeffectively treat the subject.

The pharmaceutical compositions of the invention may further compriseother bioactive agents, such as, e.g., non-Factor VII-related coagulantsor anticoagulants.

EXPERIMENTALS General Methods

α-xylosidase Assay

The α-xylosidase assays are conducted in an appropriate buffer, e.g. 50mM sodium acetate, pH 4.5, containing a suitable substrate, e.g. theO-glycopeptides that can be obtained from the O-glycopeptide map of therelevant glycoprotein (e.g., rFVIIa). The reaction is stopped after anappropriate time that can be determined experimentally, by e.g. additionof trifluoroacetic acid, and the assay mixtures are analysed by HPLC.

α-Xylosyltransferase Assay

The α-xylosyltransferase assays are conducted in an appropriate buffer,e.g. 10 mM Hepes, pH 7.2, 0.1% Triton X-100, 0.5 mM UDP-Xylose (SigmaU5875), containing a suitable substrate, e.g. the O-glycopeptides thatcan be obtained from the O-glycopeptide map of the relevant glycoprotein(e.g., rFVIIa) or the pyridyl-aminated oligosaccharides prepared asdescribed in Minamida et al. (Minamida et. al., Detection ofUDP-D-xylose: α-D-xyloside α1-3xylosyltransferase activity in humanhepatoma cell line HepG2. J. Biochem. 120 1002-1006, 1996). The reactionis stopped after an appropriate time, that can be determinedexperimentally, by e.g. addition of trifluoroacetic acid, and the assaymixtures are analysed by HPLC.

The α-xylosidase and α-xylosyltransferase assays are optimized for timeand, optionally for temperature and pH.

O-Glucosyltransferase Assay.

The O-glucosyltransferase assays are conducted, e.g., as described byShao et al. (Glycobiology 12(11) 763-770 2002).

Factor VII Assays

A suitable assay for testing for factor VIIa activity and therebyselecting suitable factor VIIa variants can be performed as a simplepreliminary in vitro test. The assay is also suitable for selectingsuitable factor VIIa variants.

In Vitro Hydrolysis Assay

Native (wild-type) factor VIIa and factor VIIa variant (both hereafterreferred to as “factor VIIa”) may be assayed for specific activities.They may also be assayed in parallel to directly compare their specificactivities. The assay is carried out in a microtiter plate (MaxiSorp,Nunc, Denmark). The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide(S-2288, Chromogenix, Sweden), final concentration 1 mM, is added tofactor VIIa (final concentration 100 nM) in 50 mM Hepes, pH 7.4,containing 0.1 M NaCl, 5 mM CaCl₂ and 1 mg/ml bovine serum albumin. Theabsorbance at 405 nm is measured continuously in a SpectraMax™ 340 platereader (Molecular Devices, USA). The absorbance developed during a20-minute incubation, after subtraction of the absorbance in a blankwell containing no enzyme, is used to calculate the ratio between theactivities of variant and wild-type factor VIIa:

Ratio=(A _(405 nm) factor VIIa variant)/(A _(405 nm) factor VIIawild-type).

Based thereon, factor VIIa variants with an activity comparable to orhigher than native factor VIIa may be identified, such as, for example,variants where the ratio between the activity of the variant and theactivity of native factor VII (wild-type FVII) is around, versus above1.0.

The activity of factor VIIa or factor VIIa variants may also be measuredusing a physiological substrate such as factor X, suitably at aconcentration of 100-1000 nM, where the factor Xa generated is measuredafter the addition of a suitable chromogenic substrate (eg. S-2765). Inaddition, the activity assay may be run at physiological temperature.

In Vitro Proteolysis Assay

Native (wild-type) Factor VIIa and Factor VIIa variant (both hereafterreferred to as “Factor VIIa”) are assayed in parallel to directlycompare their specific activities. The assay is carried out in amicrotiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) andFactor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1M NaCl, 5 mM CaCl₂ and 1 mg/ml bovine serum albumin, are incubated for15 min. Factor X cleavage is then stopped by the addition of 50 microL50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and 1 mg/mlbovine serum albumin. The amount of Factor Xa generated is measured byaddition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide(S-2765, Chromogenix, Sweden), final concentration 0.5 mM. Theabsorbance at 405 nm is measured continuously in a SpectraMax™ 340 platereader (Molecular Devices, USA). The absorbance developed during 10minutes, after subtraction of the absorbance in a blank well containingno FVIIa, is used to calculate the ratio between the proteolyticactivities of variant and wild-type Factor VIIa:

Ratio=(A405 nm Factor VIIa variant)/(A405 nm Factor VIIa wild-type).

Based thereon, factor VIIa variants with an activity comparable to orhigher than native factor VIIa may be identified, such as, for example,variants where the ratio between the activity of the variant and theactivity of native factor VII (wild-type FVII) is around 1, versus above1.0.

Thrombin Generation Assay:

The ability of factor VII or factor VII-related polypeptides (e.g.,variants) to generate thrombin can be measured in an assay comprisingall relevant coagulation factors and inhibitors at physiologicalconcentrations and activated platelets (as described on p. 543 in Monroeet al. (1997) Brit. J. Haematol. 99, 542-547 which is herebyincorporated as reference).

Clot Assays.

1st Generation Assay

The activity of the Factor VII polypeptides may also be measured using aone-stage clot assay essentially as described in WO 92/15686 or U.S.Pat. No. 5,997,864. Briefly, the sample to be tested is diluted in 50 mMTris (pH 7.5), 0.1% BSA and 100 μL is incubated with 100 μL of FactorVII deficient plasma and 200 μL of thromboplastin C containing 10 mMCa2+. Clotting times are measured and compared to a standard curve usinga reference standard or a pool of citrated normal human plasma in serialdilution.

2nd Generation Assay:

Essentially the same, except that recombinant human tissue factor isused instead for thromboplastin C.

Factor IX Assay Test for Factor IX Activity:

Suitable assays for testing for factor IX activity, and therebyproviding means for selecting suitable factor IX variants for use in thepresent invention, can be performed as simple in vitro tests asdescribed, for example, in Wagenvoord et al., Haemostasis 1990;20(5):276-88

Factor IX biological activity may also be quantified by measuring theability of a preparation to correct the clotting time of factorIX-deficient plasma, e.g., as described in Nilsson et al., 1959.(Nilsson I M, Blombaeck M, Thilen A, von Francken I., Carriers ofhaemophilia A—A laboratory study, Acta Med Scan 1959; 165:357). In thisassay, biological activity is expressed as units/ml plasma (1 unitcorresponds to the amount of FIX present in normal pooled plasma.

EXAMPLES

The following examples are intended as non-limiting illustrations of thepresent invention.

Example 1 Preparation of α-Xylosidase by Extraction and Purification

The enzyme, α-xylosidase, can be prepared from various sources, e.g.from plant material as described by Monroe et al. (Plant Physiology andBiochemistry 41:877-885 (2003)). For example, plant tissues from e.g.Arabidopsis thaliana are ground in a mortar and pestle with quartz sandin two volumes of Buffer A (40 mM Hepes, pH 7.0, 1 M NaCl), and thefiltered extract is centrifuged at 15000×g for 15 min. Ammonium sulfateis added to for example 80% saturation. Precipated proteins arecollected by centrifugation at 15000×g for 15 min and redissolved inBuffer A. The α-xylosidase is purified by chromatography, for example ona Concanavalin A-Sepharose column, on an anion-exchange column and/or onother chromatographic columns known for the skilled person. Fractionsare collected during elution and the fractions containing theα-xylosidase enzyme are identified by use of the α-xylosidase assay.

Example 2 Preparation of α-Xylosidase by Cloning and Expression in E.coli and Purification

Genes encoding α-xylosidases, which can hydrolyse alpha xylosidic bonds,have been cloned and characterized previously and genes showingsignificant homology to characterized α-xylosidases have been annotatedin the genomes from several prokaryotic and eukaryotic organisms. Thegene sequences are available in databases such as SWISS-PROT or NCBI andcan be amplified by PCR from genomic DNA from the respective organisms.Several candidates were chosen for cloning and expression in E. coliafter searching protein databases for the presence of α-xylosidaseproteins. The following candidate genes were selected on the basis ofalready existing annotation in databases (A), previous publishedcharacterization(P) or based on homology analysis to knownaxylosidases(H): gene tm0308 (Thermotoga maritima: A); gene bt3085 (2139bp) and gene bt3659(2475 bp) (Bacteroides thetaiotaomicron: A); genebf0551 (2238 bp) and gene bf1247 (2538 bp) (Bacteroides fragilis: A);gene bl02681(2310 bp)(Bacillus licheniformis: H); gene bh1905 (2328bp)(Bacillus halodurans: H), gene xylS (2196 bp)(Sulfolobussolfataricus: P); gene yicI (2319 bp) (Escherichia coli: P)

Strategy for Cloning and Expression of α-Xylosidases in E. coli

The SignalP software (Bendtsen, J. D. et al. J. Mol. Biol., 340:783-795,2004) is used to evaluate whether a signal peptide is potentiallypresent in the N-terminal of the candidate enzymes. BF0551, BF1247,BT3085, BT3659 are presumably secreted as indicated by a strongprediction of a signal peptidase I cleavage site. A methionine codonencoding a start-methionine is included in front of the first amino acidfollowing the predicted cleavage site.

Purified genomic DNA from Bacteroides thetaiotaomicron (ATCC 29148D),Bacteroides fragilis (ATCC 25285D), Bacillus haludurans (ATCC21591D&BAA-125D), Sulfolobus solfataricus (ATCC 35092D), Thermotogamaritima (ATCC 43589D) is obtained from American Type CultureCollection. In case of E. coli (strain K-12 derivative) and Bacilluslicheniformis (ATCC 28450), genomic DNA is prepared from bacterial cellscultivated overnight in LB medium using the DNeasy tissue kit (Qiagen)according to the manufactures instructions.

Forward and reverse primers for PCR amplification are designed with anextension in the 5′-ends comprising the restriction enzyme cleavagesites NdeI (or XbaI) and XmaI, respectively. PCR is performed using thefollowing conditions: 1) 95° C. for 3 min: denaturation, 2) 94° C. for30 sec: denaturation, 3) 55° C. or 60° C. for 30 sec: annealing, 4) 72°C. for 2 min: elongation. Step 2-4 is repeated for 15 cycles. PCRproducts are separated on 1% ethidium bromide agarose gels and bandsshowing the correct predicted sizes are excised from the gels andpurified using the GFX DNA purification kit (Amersham Pharmacia).Purified PCR products are cloned into the pCR2.1TOPO vector according tothe instructions of the manufacturer (Invitrogen). Clones showing thecorrect restriction enzyme cleavage profile are sequenced to evaluatethe DNA sequence. The insert representing the α-xylosidase genes arereleased from the pCR2.1TOPO vector using the relevant restrictionenzymes. A pET11a E. coli expression vector (Novagen) containing a NdeI(and XbaI) and a XmaI site is cleaved with relevant restriction enzymesand the vector part is purified as described for the PCR products.Vector and inserts are ligated together using the Rapid Ligation Kit(Roche) according to the manufacturer's instructions.

Ligation products are transformed into E. coli TOP10 (Invitrogen) cellsby means of chemical transformation or heat shock methods known to theskilled persons. Cells are plated on LB/ampecillin(Amp)-medium cultureplates overnight. Single colonies are selected from plates and grownovernight in LB/Amp medium. Purified pET plasmids from each colony arescreened for the presence of correct inserts using restriction cleavageenzymes and evaluation of sizes of released inserts.

E. Coli Rosetta DE3 (Novagen) is transformed with pET plasmidscontaining the axylosidase genes and plated on chloramphinicol(Cam)/AmpLB plates. Cells from overnight plates are resuspended in liquid Cam/AmpLB medium and diluted to OD₆₀₀=0.1. Cells in liquid medium arepropagated until OD₆₀₀=0.4-0.8. Cells are then equilibrated to atemperature of 18° C. for 30 min. and protein induction is induced with0.5 mM IPTG o/n at 18° C. Cells are harvested and pellets arere-suspended in a buffer (e.g., 25 mM Tris HCl pH 7 or 10 mM potassiumphosphate buffer pH 7) to a cell density corresponding to OD₆₀₀=˜10.Cells are sonicated on ice for 3-7 times 15-30 sec with interruptions of30 sec on ice. Cell debris is removed by centrifugation and supernatantsare assayed for activity.

Assay for α-Xylosidase Activity

Supernatants resulting from sonication are evaluated on p-nitrophenylα-D xylopyranoside (Sigma) for presence of α-xylosidase activity. Crudeenzyme is incubated with 5 mM p-nitrophenyl α-D xylopyranoside at 37° C.for 1-2 hours in a buffer (e.g., 10 mM potassium buffer pH 7 or a 25 mMTris HCl pH 7 buffer). Crude enzymes are also assayed on a fragment ofhuman FVII comprising the Xyl-Xyl-Glc-O-Ser52 glycosylation (peptidefragment consisting of amino acid residues 39-62 of FVII) to evaluatewhether the enzyme can cleave the alpha-1,3 xylosidic bonds. Theincubation with peptide is performed for 3 hours or overnight at 37° C.Peptide samples incubated with or without axylosidase are then evaluatedby MALDI MS directly after incubation to evaluate whether the enzyme canremove zero, one or two xylose sugars from the glycopeptide.

Purification of α-Xylosidases

A partial purification of the expressed α-xylosidase is performed priorto incubation with rFVII. Supernatants (from approximately 20-50 ml cellculture) obtained after cell disruption in a suitable buffer (eg. a 10mM phosphate buffer pH 7). In case of enzymes coming from thermophiles(eg. tm0308, BH1905, XylS), supernatants are also heated at 50-70° C.for 30 min, cooled on ice for 10 min and precipitate is removed bycentrifugation for 15 min at 15.000 G in order to remove thermo-labileE. coli contaminants.

The supernatants are sterile filtrated and applied to a 1 ml DEAE FFcolumn (Amersham Pharmacia). The purification is performed with the AKTAexplorer (Amersham Pharmacia) FPLC with the following buffers: Buffer A:25 mM sodium phosphate pH 7, Buffer B: 25 mM sodium phosphate pH 7 and 1M NaCl. After the application is loaded, unbound sample is washed outwith buffer A for 5 CV. A gradient from 0-100% buffer B is used for 20CV during which the target protein is eluted in fractions. Afterpurification, fractions comprising the main peak in the resultingchromatogram are assayed by incubation on p-nitrophenyl α-Dxylopyranoside or by SDS PAGE. The fractions containing the axylosidaseactivity are diluted in a 20 mM Tris HCl pH 7, 2 mM CaCl₂ buffer andconcentrated on Vivaspin 20 50.000 MWCO columns (Vivascience) bycentrifugation at 2900 rpm.

O-Glycoforms of rFVIIa with Exclusively Glucose at Serine 52

The O-glycoforms of rFVIIa with exclusively glucose at serine 52 areobtained by incubation of rFVIIa in an appropriate buffer, e.g.glycylglycine or 20 mM Tris HCl pH 7.0, 2 mM CaCl₂, with purifiedα-xylosidase for an appropriate time, that can be determinedexperimentally. Mass spectra visualizing the deglycosylation areobtained by analysing rFVII α-xylosidase incubations ESI-MS (Q-STAR).

The resulting glycan-remodeled rFVIIa is purified from the α-xylosidaseenzyme by for example anion-exchange chromatography or gel filtration orsuitable combinations. The purity of the prepared O-glycoform of rFVIIais verified by the O-glycopeptide map of rFVIIa.

Example 3 Preparation of α-Xylosidase by Cloning and Expression of T.maritima Putative α-Xylosidase Gene (tm0308) in E. coli and Purification

The above strategy (see Example 2) was followed all the way to aconclusion for tm0308. The T. maritima putative α-xylosidase gene(tm0308) was PCR amplified and cloned into a E. coli pET11a vector.Soluble tm0308 could be obtained after expression in an E. coli Rosetta(DE3) expression strain and evaluation of a crude TM0308 preparation ona p-nitrophenyl α-D xylopyranoside, clearly indicated α-xylosidaseactivity. The α-xylosidase was partly purified using DEAE FFchromatography followed by up-concentration by ultra filtration. Thepartly purified enzyme was incubated with FVII in a 25 mM Tris pH 7, 2mM CaCl₂ buffer at different enzyme/FVII ratios for 3 hours at 50° C. orovernight at 37° C. Controls with identical compositions of α-xylosidaseand rFVII, to which synthetic substrate was added, showed that theenzyme was active under these conditions and it was possible tovisualize FVII with and without xylosidase treatment on SDS-gels and byESI-MS. However, no significant removal of the xylose sugars linked toGlc-O-Ser52 could be detected in this first experiment. In contrast,removal of xylose from a purified reduced and alkylated FVIIa peptidecomprising Xyl-Xyl-Glc-O-Ser52 was observed.

Example 4 Preparation of α-Xylosidase by Cloning and Expression in E.coli

The following constructs have been cloned into the pET expressionvectors in accordance with the strategy described in Example 2: Genebl2681(2310 bp)(Bacillus licheniformis: H); gene bl1905 (2328bp)(Bacillus halodurans: H), gene xylS (2196 bp)(Sulfolobussolfataricus: P); gene yicI (2319 bp) (Escherichia coli: P).

The constructs will be expressed in Rosetta, isolated, purified, andevaluated for α-xylosidase activity in accordance with theabove-described strategy.Each α-xylosidase will be incubated with rFVIIa in an appropriatebuffer, e.g. glycylglycine or 20 mM Tris HCl pH 7.0, 2 mM CaCl₂, for anappropriate time that can be determined experimentally and MS Spectravisualizing the deglycosylation will be obtained by analysing rFVIIα-xylosidase incubations ESI-LC-MS (Q-STAR).

The resulting glycan-remodeled rFVIIa will be purified from theα-xylosidase enzyme by for example anion-exchange chromatography or gelfiltration or suitable combinations thereof. The purity of the preparedO-glycoform of rFVIIa is verified by the O-glycopeptide map of rFVIIa.

Example 5 Preparation of Truncated α-Xylosidase by Cloning andExpression in E. coli

The crystal structure of YicI was recently solved. Thus, cloning of atruncated α-xylosidase enzyme representing an active, catalytical domainof the YicI protein (or other similar α-xylosidases) may be possible andis being planned, since a smaller enzyme, if active, may better accessthe Xyl-Xyl-Glc-O-Ser52 present in native rFVIIa. A domain comprisingthe active site in the enzyme is predicted from the structure. Genesequence encoding this part of the YicI sequence is prepared from thealready existing YicI pET11a plasmid for an example by PCR amplificationof relevant areas of the YicI gene, The primers used for PCR will haveextensions with restriction enzyme sites that can be used for ligationof the truncated YicI gene into the pET11a vector. The truncated enzymewill after expression and purification be evaluated for its potentialfor deglycosylation of rFVIIa as described above.

Example 6 Preparation of rFVIIa with Exclusively Xylose-Glucose atSerine 52 or Exclusively Xylosexylose-Glucose at Serine 52 byα-Xylosyltransferase Treatment Preparation of α-Xylosyltransferase

The enzyme, UDP-D-xylose: β-D-glucoside α-1,3-D-xylosyltransferase, canbe prepared from HepG2 cells as described by Omichi et al. (1997). Inshort, HepG2 cells are grown in a medium supplemented with 10% fetalcalf serum. The microsomal fraction is prepared by homogenisation of thecells followed by centrifugation. The α-xylosyltransferase enzyme ispurified by chromatography, for example on an anion-exchange columnand/or on other chromatographic columns known for the skilled person.Fractions are collected during elution and the fractions containing theα-xylosyltransferase enzyme are identified by use of theα-xylosyltransferase assay.

The enzyme, UDP-D-xylose: β-D-xyloside α1,3-xylosyltransferase, can beprepared from HepG2 cells as described by Minamida et al. (1996). Inshort, HepG2 cells are grown in a medium supplemented 10% fetal calfserum. The microsomal fraction is pre-pared by homogenisation of thecells followed by centrifugation. The α-xylosyltransferase enzyme ispurified by chromatography, for example on an anion-exchange columnand/or on other chromatographic columns known for the skilled person.Fractions are collected during elution and the fractions containing theα-xylosyltransferase enzyme are identified by use of theα-xylosyltransferase assay.

α-Xylosyltransferase Assay

The α-xylosyltransferase assays are conducted in an appropriate buffer,e.g. 10 mM Hepes, pH 7.2, 0.1% Triton X-100, 0.5 mM UDP-Xylose (SigmaU5875), containing a suitable substrate, e.g. the O-glycopeptides thatcan be obtained from the O-glycopeptide map of rFVIIa or thepyridylaminated oligosaccharides prepared as described in Minamida etal. (Minamida et. al., Detection of UDP-D-xylose: α-D-xylosideα1-3xylosyltransferase activity in human hepatoma cell line HepG2. J.Biochem. 120 1002-1006, 1996). The reaction is stopped after anappropriate time, that can be determined experimentally, by e.g.addition of trifluoroacetic acid, and the assay mixtures are analysed byHPLC.

O-Glycoforms of rFVIIa with Exclusively Xylose-Glucose- at Serine 52

The O-glycoforms of rFVIIa with exclusively xylose-glucose at serine 52are obtained by (1) treatment of rFVIIa with xylosidase as describedabove, (2) purification of the xylosidase treated rFVIIa from thexylosidase by for example anion-exchange chromatography, and (3) byincubation of xylosidase-treated rFVIIa in an appropriate buffer, e.g.glycylglycine, pH 7.0, 10 mM calcium chloride, with purifiedUDP-D-xylose: β-D-glucoside α-1,3-D-xylosyltransferase and UDP-D-xylosefor an appropriate time, that can be determined experimentally. Theresulting glyco-remodelled rFVIIa is purified from the UDP-D-xylose:β-D-glucoside α-1,3-D-xylosyltransferase enzyme by for exampleanion-exchange chromatography. The purity of the prepared O-glycoform ofrFVIIa is verified by the O-glycopeptide map of rFVIIa.

O-Glycoforms of rFVIIa with Exclusively Xylose-Xylose-Glucose- at Serine52

The O-glycoforms of with xylose-xylose-glucose at serine 52 are obtainedby (1) treatment of rFVIIa with xylosidase as described above, (2)purification of the xylosidase treated rFVIIa from the xylosidase by forexample anion-exchange chromatography, (3) further treatment withUDP-D-xylose: β-D-glucoside α-1,3-D-xylosyltransferase and UDP-D-xyloseas described above, and (4) by incubation of the product in anappropriate buffer, e.g. glycylglycine, pH 7.0, 10 mM calcium chloride,with purified UDP-D-xylose: α-D-xyloside α1,3-xylosyltransferase andUDP-D-xylose for an appropriate time, that can be determinedexperimentally. The resulting glyco-remodelled rFVIIa is purified fromthe UDP-D-xylose: α-D-xyloside α1,3-xylosyltransferase enzyme by forexample anion-exchange chromatography. The purity of the preparedO-glycoform of rFVIIa is verified by the O-glycopeptide map of rFVIIa.

Example 7 Analysis of O-Glycoform Pattern of rFVIIa

Tryptic Peptide Mapping of the rFVIIa Light Chain

The relative content of the O-glycoforms of rFVIIa is determined bytryptic peptide mapping of the rFVIIa light chain. The rFVIIa is reducedand alkylated and the rFVIIa light chain is purified on a RP-HPLC columneluted with an acetonitrile gradient in water:trifluoroacetic acid. Thepurified rFVIIa light chain is buffer-exchanged to Tris buffer, pH 7.5and digested with trypsin. The tryptic digest of the rFVIIa light chainis analysed on a RP-HPLC column (for example Nucleosil C18, 5μ, 300 Å,4.0×250 mm, Macherey-Nagel 720065) eluted with an acetonitrile gradient(0%-45% acetonitrile in 100 min) in water: trifluoroacetic acid (seeFIG. 2). Flow is 1.0 ml/min and detection is UV at 215 nm.

The peaks containing the O-glycopeptides of rFVIIa are eluted afterapprox. 60-65 min where the 1st and the 3rd peak contain O-glycopeptideswith a xylose-xylose-glucose-linked to serine 52, and the 2nd and 4thpeak contain O-glycopeptides with a glucose linked to serine 52.

Similarly, the 1st and the 2nd peak contain O-glycopeptides with atetrasacharide linked to serine 60, and the 3rd and the 4th peak containO-glycopeptides with a fucose linked to serine 60.

Tryptic Peptide Mapping of rFVIIa

The O-glycoform pattern can be analysed by tryptic peptide mapping ofrFVIIa. The rFVIIa is buffer-exchanged to Tris buffer, pH 7.5, anddigested with trypsin. The tryptic digest of the rFVIIa is analysed on aRP-HPLC column (for example Nucleosil C18, 5μ, 300 Å, 4.0×250 mm,Macherey-Nagel 720065) eluted with an acetonitrile gradient (0%-45%acetonitrile in 100 min) in water: trifluoroacetic acid. Flow is 1.0ml/min and detection is UV at 215 nm.

The peaks containing the O-glycopeptides of rFVIIa are eluted afterapprox. 67-70 min where the 1st peak contains O-glycopeptides with axylose-xylose-glucose linked to serine 52, and the 2nd peak containsO-glycopeptides with a glucose linked to serine 52.

Total Mass Analysis of rFVIIa

The O-glycoform pattern can be analysed by total mass analysis ofrFVIIa. The rFVIIa is desalted on a Millipore ZipTip C4 columnequilibrated with 0.1% formic acid and eluted with 3% formic acid in 90%methanol. The eluted sample is analysed by the nanospray technique on aQstar XL mass spectrometer.

The major peak at approximately 50500 Da represents rFVIIa O-glycoformswith a glucose linked to serine 52 and the major peak at approximately50800 Da represents rFVIIa O-glycoforms with a xylose-xylose-glucoselinked to serine 52.

Example 8 Purification of Glc-O-Ser52-FVII and Xyl-Xyl-Glc-O-Ser52-FVII

Glc-O-ser52-FVII and Xyl-Xyl-Glc-O-Ser52-FVII was purified using twocycles of hydrophobic interaction chromatography (HIC). The column (1.0cm in inner diameter×7.0 cm length=5.5 ml column volume (CV)) packedwith Toso Haas TSK-Gel phenyl 5 PW, was equilibrated with 5 CV 10 mMhistidine, 10 mM CaCl2, 2.0 M NH4-acetate, pH 6.0. The column was loadedwith approximately 2.5 mg of FVII pr. ml resin. To the load solution 2.0M NH4-acetate and 10 mM CaCl2 was added prior to load. The column waswashed with 5 CV 10 mM histidine, 10 mM CaCl₂, 2.0 M NH4-acetate, pH6.0. Elution was performed using a 20 CV linear gradient from 10 mMhistidine, 10 mM CaCl2, 2.0 M NH4-acetate, pH 6.0 to 10 mM histidine, 10mM CaCl2, pH 6.0. The purification was performed at a flow rate of 6CV/h and at a temperature of 5° C. Fractions were collected duringelution.

The FVII eluted in two overlapping major peaks (see FIG. 4: Chromatogramfrom first HIC cycle). Fractions containing the first peak were pooled(fraction “A”, FIG. 4) and further purified by a second cycle of HIC,using the same chromatographic procedure as for the first HIC cycle (seeFIG. 5: Chromatogram obtained by reloading fraction “A” onto the HICcolumn). Fractions containing the second major peak (fraction “B”, FIG.4) were pooled as well and further purified by a second cycle of HIC,using the same chromatographic procedure as for the first HIC cycle (seeFIG. 6: Chromatogram obtained by reloading fraction “B” onto the HICcolumn).

Purified Glc-O-Ser52-FVII was identified in the peak fraction, fraction10 (FIG. 5), obtained by reloading fraction “A” onto the second HICstep. Purified Xyl-Xyl-Glc-O-Ser52-FVII was identified in the peakfraction, fraction 15 (FIG. 6), obtained by reloading fraction “B” ontothe second HIC step. The identification was obtained by tryptic peptidemapping of rFVIIa as described in Example 7 (FIGS. 7A and 7B) and bytotal mass analysis of rFVIIa as described in Example 7 (FIGS. 8A and8B). Both analyses showed a high content of Glc-O-Ser52-rFVIIa and a lowcontent of Xyl-Xyl-Glc-O-Ser52-rFVIIa in the peak fraction, Fraction 10,and a low content of Glc-O-Ser52-rFVIIa and a high content ofXyl-Xyl-Glc-O-Ser52-rFVIIa in the peak fraction, Fraction 15. Aquantitation of the content of the O-glycoforms in the two peakfractions could not be obtained due to relatively low rFVIIa content inthe fractions (FIGS. 7A and 7B: Tryptic peptide mapping: Other peptidefragments of rFVIIa co-eluted with or eluted close to theO-glycopeptides, and the content of O-glycopeptides in low amounts couldtherefore not be determined.) (FIGS. 8A and 8B: Total mass analysis:Other O- and/or N-glycoforms of rFVIIa, for example N-glycoforms ofrFVIIa lacking one N-acetylneuraminic acid, appeared in the massspectra, and the content of O-glycoforms of rFVIIa in low amounts couldtherefore not be determined).

The specific activities of the peak fractions obtained from the HIC(Table 1) were determined by the 1st generation clotting assay. It wasfound that the Glc-O-Ser52-rFVIIa O-glycoform had a low specificactivity while the Xyl-Xyl-Glc-O-Ser52-rFVIIa O-glycoform had a highspecific activity.

TABLE 1 Specific activities determined using the 1st generation clottingassay for the peak fractions obtained from HIC. The content of rFVIIawas determined by HPLC. Sample Specific activity PS5002-014 Frak. 10 44IU/μg PS5002-015 Frak. 15 61 IU/μg PS5002-014/015 starting material 53IU/μg

Example 9 Purification by Hydrophobic Interaction Chromatography

Highly purified Glc-O-Ser52-rFVIIa preparations and highly purifiedXyl-Xyl-Glc-O-Ser52-rFVIIa preparations can be obtained by repeatedpurification on the hydrophobic interaction chromatography as describedabove. Highly purified Glc-O-Ser52-rFVIIa and Xyl-Xyl-Glc-O-Ser52-rFVIIapreparations with higher rFVIIa content can be obtained by increasingthe amount of starting material for the hydrophobic interactionchromatography performed as described above. The content of eachO-glycoform of rFVIIa in the highly purified preparations with higherrFVIIa content can be quantitated by tryptic peptide mapping of therFVIIa light chain as described in Example 7. The specific activities ofthe highly purified preparations can be determined by the 1st generationclotting assay as above.

1. A preparation of a glycoprotein containing aCys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is linkedto a sugar chain by a Glc-O-Ser/Thr covalent bond, X1 and X2 eachrepresent any amino acid residue and the preparation has a substantiallyuniform serine/threonine-linked glycosylation pattern.
 2. Thepreparation according to claim 1, wherein the glycosylation pattern isat least 80% uniform.
 3. The preparation according to claim 1, whereinthe serine/threonine-linked sugar chain is Xyl-Xyl-Glc-.
 4. Thepreparation according to claim 1, wherein the serine/threonine-linkedsugar chain is Xyl-Glc-.
 5. The preparation according to claim 1,wherein the serine/threonine-linked sugar chain is Glc-.
 6. Thepreparation according to claim 1, wherein the glycoprotein is selectedfrom the group consisting of Factor VII polypeptides, Factor VII-relatedpolypeptides, Factor IX polypeptides, Factor IX-related polypeptides,Factor X polypeptides, Factor X-related polypeptides, Factor XIIpolypeptides, and protein Z polypeptides.
 7. The preparation accordingto claim 6, wherein the glycoprotein is human Factor VII.
 8. Apreparation according to claim 6, wherein the glycoprotein is a variantof Factor VII and wherein the ratio between the activity of the FactorVII-variant and the activity of native human factor VIIa (wild-typeFVIIa) is at least about 1.25 when tested in the “In Vitro HydrolysisAssay” or in the “In vitro Proteolysis Assay”.
 9. The preparationaccording to claim 6, wherein the glycoprotein is human Factor IX or ahuman Factor IX sequence variant.
 10. A method for making a preparationaccording to claim 5 comprising: (a) obtaining a preparation of aprecursor glycoprotein containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif,wherein the serine/threonine is linked to a sugar chain by Glc-O-Ser/Thrcovalent bond and X1 and X2 each represent any amino acid residue; and(b) contacting the preparation obtained in step (a) with an α-xylosidaseunder conditions appropriate for removing xylose residues from theprecursor glycoprotein, thereby producing the glycoprotein.
 11. Themethod according to claim 10, further including the step of isolatingthe glycoprotein prepared in step (b).
 12. The method according to claim10, wherein the glycosylation is a serine glycosylation.
 13. The methodaccording to claim 10 10, further including the step of analysing thestructure of the sugar chain linked to the glycoprotein to determine aglycoform pattern, and, optionally, repeating step (b) until the desiredglycoform pattern is achieved.
 14. A method for making a preparationaccording to in claim 6 comprising: (a) obtaining a preparation of apolypeptide containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein X1 andX2 each represent any amino acid residue; and (b) contacting thepreparation obtained in step (a) with a O-glucosyltransferase and anactivated glucose donor under conditions appropriate for transferring aglucose residue from the glucose donor moiety to the serine/threoninethereby producing the glycoprotein.
 15. The method according to claim14, further including the step of isolating the glycoprotein prepared instep (b).
 16. The method according to claim 14, wherein theglycosylation is a serine glycosylation.
 17. The method according toclaim 14, further including the step of analyzing the structure of thesugar chain linked to glycoproteins in the preparation to determine ifthe glycoproteins have a desired glycoform pattern, and, optionally,repeating step (b) until the desired glycoform pattern is achieved. 18.A method for making a preparation according to claim 4 comprising: (a)obtaining a preparation of a precursor glycoprotein containing aCys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is linkedto a sugar chain by a Glc-O-Ser/Thr covalent bond and X1 and X2 eachrepresent any amino acid residue; and (b) contacting the preparationobtained in step (a) with (i) UDP-D-xylose: β-D-glucosideα-1,3-D-xylosyltransferase and (ii) an activated xylosyl donor underconditions appropriate for transferring a xylose residue from an xylosedonor moiety to an acceptor moiety, thereby producing the glycoprotein.19. The method according to claim 18, further including the step ofisolating the glycoprotein prepared in step (b).
 20. The methodaccording to claim 18, wherein the glycosylation is a serineglycosylation.
 21. The method according to claim 18, further includingthe step of analyzing the structure of the sugar chain linked toglycoproteins in the preparation to determine if the glycoproteins havea desired glycoform pattern, and, optionally, repeating step (b) untilthe desired glycoform pattern is achieved.
 22. The method according toclaim 18, further including the step of removing terminalxylose-residues by subjecting the preparation obtained in step (a) tothe method described in claim 10 prior to step (b).
 23. A method formaking a preparation according to claim 3 comprising: (a) obtaining apreparation of a precursor glycoprotein containing aCys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is linkedto a sugar chain by a Glc-O-Ser/Thr covalent bond and X1 and X2 eachrepresent any amino acid residue; (b) contacting the preparationobtained in step (a) with UDP-D-xylose: β-D-glucosideα-1,3-D-xylosyltransferase and an activated xylosyl donor underconditions appropriate for transferring a xylose residue from a xylosedonor moiety to an acceptor moiety; and (c) contacting the preparationobtained in step (b) with UDP-D-xylose: α-D-xylosideα-1,3-xylosyltransferase and an activated xylosyl donor under conditionsappropriate for transferring a xylose residue from a xylose donor moietyto an acceptor moiety, thereby producing the glycoprotein.
 24. Themethod according to claim 23, further including the step of isolatingthe preparation obtained in step (b) prior to subjecting the preparationto step (c).
 25. The method according to claim 23, further including thestep of isolating the glycoprotein prepared in step (c).
 26. The methodaccording to claim 23, wherein the glycosylation is a serineglycosylation.
 27. The method according to claim 23, further includingthe step of analyzing the structure of the sugar chain linked to theglycoproteins in the preparation to determine if the glycoproteins havea desired glycoform pattern, and, optionally, repeating step (b) and/orstep (c) until the desired glycoform pattern is achieved.
 28. The methodaccording to claim 23, further including the step of removing terminalxylose-residues by subjecting the preparation obtained in step (a) tothe method described in claim 10 prior to step (b).
 29. The preparationaccording to claim 2, wherein the glycosylation pattern is at least 90%uniform.
 30. The preparation according to claim 29, wherein theglycosylation pattern is at least 98% uniform.